Acetylated sialic acid residues and blood group antigens localise within the epithelium in microvillous atrophy indicating internal accumulation of the glycocalyx

PubMed Central

Phillips, A D; Brown, A; Hicks, S; Schüller, S; Murch, S H; Walker-Smith, J A; Swallow, D M

2004-01-01

Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition. Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically. Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral (PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules. Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material. PMID:15542511

A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

PubMed

Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

2012-12-01

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.

Skeletal advance and arrest in giant non-metamorphosing African clawed frog tadpoles (Xenopus laevis: Daudin)

PubMed Central

Kerney, Ryan; Wassersug, Richard; Hall, Brian K

2010-01-01

This study examines the skeletons of giant non-metamorphosing (GNM) Xenopus laevis tadpoles, which arrest their development indefinitely before metamorphosis, and grow to excessively large sizes in the absence of detectable thyroid glands. Cartilage growth is isometric; however, chondrocyte size is smaller in GNM tadpoles than in controls. Most cartilages stain weakly with alcian blue, and several cartilages are calcified (unlike controls). However, cartilages subjacent to periosteum-derived bone retain strong affinities for alcian blue, indicating a role for periosteum-derived bone in the retention of glycosaminoglycans during protracted larval growth. Bone formation in the head, limb, and axial skeletons is advanced in comparison with stage-matched controls, but arrests at various mid-metamorphic states. Both dermal and periosteum-derived bones grow to disproportionately large sizes in comparison to controls. Additionally, mature monocuspid teeth form in several GNM tadpoles. Advances in skeletal development are attributable to the old ages and large sizes of these tadpoles, and reveal unexpected developmental potentials of the pre-metamorphic skeleton. PMID:20402828

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice.

PubMed

Oliveira, J T; Santos, T C; Martins, L; Silva, M A; Marques, A P; Castro, A G; Neves, N M; Reis, R L

2009-10-01

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright (c) 2009 John Wiley & Sons, Ltd.

[Histochemical detection of glycoproteins and glycosaminoglycans in the respiratory mucosa of albino rats during estrous cycle, pregnancy and puerperium].

PubMed

Pontes, P A; Simões, M J; Merzel, J

1989-11-01

In this work we attempted to detect, with histochemical methods, the possible modifications in the mucus of the respiratory mucosa of albino female rats during estral cycle, pregnancy and puerperium. Based on its results, it was possible to conclude that: a--There were no modifications in the nature of the epithelial and supraepithelial mucus during the studied periods: b--The Alcian Blue staining from lamina propria is absent during pregnancy and present during puerperium.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

PubMed

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

Histochemical and immunohistochemical characterization of chordoma in ferrets.

PubMed

Yui, Takeshi; Ohmachi, Tetsuo; Matsuda, Kazuya; Okamoto, Minoru; Taniyama, Hiroyuki

2015-04-01

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as "physaliphorous cells", were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a "chordoid" or "cobblestone" manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer's mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.

Locally infiltrative ameloblastic fibroma in a rhesus macaque (Macaca mulatta) with characterizations of its proliferating activity and biological behavior

PubMed Central

Liu, David X.; Doyle, Lara A.; Bouljihad, Mostafa T.; Didier, Peter J.; Gilbert, Margaret H.; Wang, Xiaolei; Pahar, Bapi; Bohm, Rudolf P.; Veazey, Ronald S.; Lackner, Andrew A.

2014-01-01

An 8-year-old male rhesus macaque (Macaca mulatta) presented with unilateral enlargement of the left mandible. Radiographs revealed a marked expansion of the left mandible with a multilocular radiolucent mass with abundant osteolysis. The mass was grossly firm, fleshy, and gelatinous on the cut surface. Histologically, the mass was locally infiltrative and composed of neoplastic epithelial and mesenchymal components that stained positive for cytokeratin and vimentin, respectively. Occasional densely spherical condensations of fibroblasts resembling the cap stage of odontogenesis were present in the mesenchyma. Immunohistochemical staining with Ki-67, S-100, and CD34 indicated that both epithelial and mesenchymal components of the neoplasm had low proliferation. Alcian blue, periodic acid–Schiff, and trichrome stains showed an immature stromal component with no collagen formation. Based on the clinical, histologic, and immunophenotypic features, the tumor was identified as a locally infiltrative ameloblastic fibroma. PMID:22529141

Immunohistochemical/histochemical double staining method in the study of the columnar metaplasia of the oesophagus.

PubMed

Cabibi, D; Giannone, A G; Mascarella, C; Guarnotta, C; Castiglia, M; Pantuso, G; Fiorentino, E

2014-03-05

Intestinal metaplasia in Barrett's oesophagus (BO) represents an important risk factor for oesophageal adenocarcinoma. Instead, few and controversial data are reported about the progression risk of columnar-lined oesophagus without intestinal metaplasia (CLO), posing an issue about its clinical management. The aim was to evaluate if some immunophenotypic changes were present in CLO independently of the presence of the goblet cells. We studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metaplasia, by performing some immunohistochemical stainings (CK7, p53, AuroraA) combined with histochemistry (Alcian-blue and Alcian/PAS), with the aim of simultaneously assess the histochemical features in cells that shows an aberrant expression of such antigens. We evidenced a cytoplasmic expression of CK7 and a nuclear expression of Aurora A and p53,  both in goblet cells of BO and in non-goblet cells of CLO, some of which showing mild dysplasia. These findings suggest that some immunophenotypic changes are present in CLO and they can precede the appearance of the goblet cells or can be present independently of them, confirming the conception of BO as the condition characterized by any extention of columnar epithelium. This is the first study in which a combined immunohistochemical/histochemical method has been applied to Barrett pathology.

Comprehensive histological evaluation of bone implants

PubMed Central

Rentsch, Claudia; Schneiders, Wolfgang; Manthey, Suzanne; Rentsch, Barbe; Rammelt, Stephan

2014-01-01

To investigate and assess bone regeneration in sheep in combination with new implant materials classical histological staining methods as well as immunohistochemistry may provide additional information to standard radiographs or computer tomography. Available published data of bone defect regenerations in sheep often present none or sparely labeled histological images. Repeatedly, the exact location of the sample remains unclear, detail enlargements are missing and the labeling of different tissues or cells is absent. The aim of this article is to present an overview of sample preparation, staining methods and their benefits as well as a detailed histological description of bone regeneration in the sheep tibia. General histological staining methods like hematoxylin and eosin, Masson-Goldner trichrome, Movat’s pentachrome and alcian blue were used to define new bone formation within a sheep tibia critical size defect containing a polycaprolactone-co-lactide (PCL) scaffold implanted for 3 months (n = 4). Special attention was drawn to describe the bone healing patterns down to cell level. Additionally one histological quantification method and immunohistochemical staining methods are described. PMID:24504113

Changes in the oviducal epithelium during the estrous cycle in the marsupial Monodelphis domestica

PubMed Central

Kress, Annetrudi; Morson, Gianni

2007-01-01

The Monodelphis oviduct can be divided into four anatomical segments: preampulla (comprising fimbriae and infundibulum), ampulla, isthmus with crypts and uterotubal junction. Ovaries are enclosed in a periovarial sac, the bursa, and in some specimens tubules of an epoophoron could be identified. In both structures non-ciliated cells develop small translucent vesicles, which accumulate in the cell apices and presumably produce fluid as often seen in the bursa and in the tubules of the epooophoron. These vesicles do not stain with Alcian blue or PAS. The same applies also to the non-ciliated cells of the fimbriae. The oviducal epithelium of ampulla and the surface epithelium of the isthmus consisting of ciliated and non-ciliated, secretory cells undergo considerable changes during the estrous cycle. Proestrus shows low numbers of ciliated cells, some are in the process of neo-ciliogenesis, non-ciliated cells carry solitary cilia and few remnant secretory granules from the previous cycle may be found. At estrus the amount of ciliated cells in ampulla and isthmus has increased, most non-cililated cells lost the solitary cilia, developed longer microvilli and formed numerous secretory granules in their cell apices. At postestrus secretory products, often surrounded by membranes, are extruded into the oviducal lumen and contribute towards egg coat formation. First signs of deciliation processes are apparent. Solitary cilia reappear. At metestrus only few secretory cells are left with some secretory material. The lumen is often filled with shed cilia and cell apices. Proliferation of basal bodies within non-secretory cells indicate the formation of new ciliated cells. The non-ciliated epithelial cells of the isthmic crypts form no secretory granules but accumulate a great number of translucent vesicles, which in contrast to the secretory granules do not stain with Alcian blue or PAS. PMID:17883438

Structure-matched Phthalocyanine Ion Pair as a Red-emitting Fluorescent Optical Probe for the Analysis of Sodium Dodecylbenzenesulfonate with High Specificity and Sensitivity.

PubMed

Yu, Fei; Guo, Menglin; Deng, Yabin; Lu, Yin; Chen, Lin; Huang, Ping; Li, Donghui

2016-01-01

We have found that a positively charged cationic copper phthalocyanine, Alcian blue (Alcian blue 8GX), can efficiently quench the fluorescence of an oppositely charged red fluorescent phthalocyanine compound with a matched molecular structure, tetrasulfonated aluminum phthalocyanine (AlS4Pc), because of the formation of an ion pair complex (AlS4Pc-Alcian blue 8GX) that exhibits almost no fluorescence. An investigation was carried out on the fluorescence recovery of AlS4Pc-Alcian blue 8GX caused by a series of anionic surfactants containing a sulfonic group (sodium dodecylbenzenesulfonate (SDBS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate (SDS)). The results showed that SDBS exhibited a significant response, and the highest sensitivity among the surfactants. Due to its high efficiency of fluorescence quenching and the high level of fluorescence recovery, direct observes can even be performed by the naked eye. The results revealed that the Alcian blue 8GX-AlS4Pc ion-pair complex fluorescent probe only responded to SDBS in the low-concentration range. Based on the new founding, this study proposed a novel principle and method of fluorescence enhancement to specifically measure the concentration of SDBS, thereby achieving a highly sensitive and highly specific determination of SDBS. Under the optimal conditions, the fluorescence intensity (I(f)) of the system and the concentration of SDBS in the range of 1 × 10(-7) - 1 × 10(-5) mol/dm(3) exhibited a good linear relationship. This method is highly sensitive, and the operation is simple and rapid. It had been applied for the quantitative analysis of SDBS in environmental water, while achieving satisfactory results compared with those of the standard method. This study developed a new application of the fluorescent phthalocyanine compounds used as molecular probes in analytical sciences.

Carbonate crystals precipitated by freshwater bacteria and their use as a limestone consolidant.

PubMed

Zamarreño, Dania V; Inkpen, Robert; May, Eric

2009-09-01

Bacterial carbonate precipitation is known to be a natural phenomenon associated with a wide range of bacterial species. Recently, the ability of bacteria to produce carbonates has been studied for its value in the conservation of limestone monuments and concrete. This paper describes investigations of carbonate crystals precipitated by freshwater bacteria by means of histological (Loeffler's methylene blue and alcian blue-periodic acid-Schiff stain) and fluorescence (CTC [5-cyano-2,3-ditolyl tetrazolium chloride]) stains, determination of cell viability inside carbonate crystals, and pore size reduction in limestone by image analysis. Carbonate crystals were found to be composed of bacteria embedded in a matrix of neutral and acid polysaccharides. Cell viability inside the carbonate crystals decreased with time. On stone, bacteria were found to form carbonate crystals, with only a few bacteria remaining as isolated cells or as cell aggregates. Pore size was reduced by about 50%, but no blockage was detected. Taken together, the results of this research provide some reassurance to conservators that biocalcification by bacteria could be a safe consolidation tool in a restoration strategy for building stone conservation.

Carbonate Crystals Precipitated by Freshwater Bacteria and Their Use as a Limestone Consolidant▿

PubMed Central

Zamarreño, Dania V.; Inkpen, Robert; May, Eric

2009-01-01

Bacterial carbonate precipitation is known to be a natural phenomenon associated with a wide range of bacterial species. Recently, the ability of bacteria to produce carbonates has been studied for its value in the conservation of limestone monuments and concrete. This paper describes investigations of carbonate crystals precipitated by freshwater bacteria by means of histological (Loeffler's methylene blue and alcian blue-periodic acid-Schiff stain) and fluorescence (CTC [5-cyano-2,3-ditolyl tetrazolium chloride]) stains, determination of cell viability inside carbonate crystals, and pore size reduction in limestone by image analysis. Carbonate crystals were found to be composed of bacteria embedded in a matrix of neutral and acid polysaccharides. Cell viability inside the carbonate crystals decreased with time. On stone, bacteria were found to form carbonate crystals, with only a few bacteria remaining as isolated cells or as cell aggregates. Pore size was reduced by about 50%, but no blockage was detected. Taken together, the results of this research provide some reassurance to conservators that biocalcification by bacteria could be a safe consolidation tool in a restoration strategy for building stone conservation. PMID:19617383

Anatomic changes due to interspecific grafting in cassava (Manihot esculenta).

PubMed

Bomfim, N; Ribeiro, D G; Nassar, N M A

2011-05-31

Cassava rootstocks of varieties UnB 201 and UnB 122 grafted with scions of Manihot fortalezensis were prepared for anatomic study. The roots were cut, stained with safranin and alcian blue, and examined microscopically, comparing them with sections taken from ungrafted roots. There was a significant decrease in number of pericyclic fibers, vascular vessels and tyloses in rootstocks. They exhibited significant larger vessels. These changes in anatomic structure are a consequence of genetic effects caused by transference of genetic material from scion to rootstock. The same ungrafted species was compared. This is the first report on anatomic changes due to grafting in cassava.

DIAZOPHTHALOCYANINS AS REAGENTS FOR FINE STRUCTURAL CYTOCHEMISTRY

PubMed Central

Tice, Lois Withrow; Barrnett, Russell J.

1965-01-01

This paper reports the synthesis of 14 diazophthalocyanins containing Mg, Cu, or Pb as the chelated metal. To assess the usefulness of these compounds for fine structural cytochemistry, the relative coupling rates with naphthols were tested as well as the solubility of the resulting azo dyes. Three of the diazotates were reacted with tissue proteins in aldehyde-fixed material, and the density increases thus produced were compared in the electron microscope with those produced by staining similarly fixed material with the phthalocyanin dye, Alcian Blue. Finally, one of the diazotates was used as a capture reagent for the demonstration of the sites of acid phosphatase activity with the electron microscope. PMID:14283629

Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs.

PubMed

Röhe, Ilen; Hüttner, Friedrich Joseph; Plendl, Johanna; Drewes, Barbara; Zentek, Jürgen

2018-02-05

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.

Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

PubMed

Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Lavarello, Chiara; Inglese, Elvira; Bruschi, Maurizio; Ghiggeri, Gian Marco; Boschetti, Egisto; Righetti, Pier Giorgio

2015-01-01

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

Metastatic iridociliary adenocarcinoma in a labrador retriever.

PubMed

Zarfoss, M K; Dubielzig, R R

2007-09-01

An enucleated left eye from a 15-year-old female spayed Labrador Retriever was received by the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) for histopathologic evaluation. Routine histologic preparation included staining with hematoxylin and eosin, and with alcian blue periodic acid-Schiff (PAS). At necropsy 9 months later, all grossly abnormal tissues (ipsilateral orbit and lung) were submitted to the COPLOW for histopathologic evaluation. Histopathologic evaluation of the globe revealed extensive invasion of the uvea and sclera by a pleomorphic cell population that formed disorganized cords and exhibited PAS-positive basement membrane material. Necropsy revealed a morphologically similar tumor in the ipsilateral orbit and lung. On immunohistochemical examination, the intraocular tumor stained diffusely immunopositive for vimentin, S-100, and neuron-specific enolase and multifocally, sparsely immunopositive for cytokeratin AE1/AE3. The orbital and thoracic tumors stained positively for vimentin but negatively for cytokeratin AE1/AE3. There are few reports of canine metastatic iridociliary adenocarcinoma in the literature; this is the first with immunohistochemical analysis.

Ultrastructure of the platypus and echidna mandibular glands.

PubMed

Krause, W J

2011-10-01

The secretory units of the platypus and echidna mandibular glands consist of a single serous cell type. Secretory granules within the cells of the platypus mandibular gland stained intensely with the periodic acid-Schiff staining procedure but failed to stain with Alcian Blue, suggesting the granules contained neutral glycoproteins. Secretory granules within the mandibular glands of the echidna failed to stain with the methods used indicating little if any glycoprotein was associated with the secretory granules. Ultrastructurally, secretory granules of the platypus mandibular gland were electron dense with a central core of less electron-dense material and were membrane bound. In contrast, those of the echidna presented a lamellated appearance and also were limited by a membrane. These secretory granules appeared to form as a result of concentric layering of lamellae within cisternae of the Golgi membranes. The intralobular ductal system of the platypus was more extensively developed than that of the echidna. The striated ducts of both species were characterized by elaborate infoldings of the basolateral plasmalemma and an abundance of associated mitochondria. © 2011 Blackwell Verlag GmbH.

Atypical myxoma.

PubMed

Hwang, J J; Lien, W P; Kuan, P; Hung, C R; How, S W

1991-08-01

We report the case of a 38-year-old woman with a large thin-walled cystic mass (6 x 5 x 4.5 cm) filled with arterial blood in the right atrium. The cystic mass with blood content was clearly delineated by transesophageal cross-sectional echocardiography and magnetic resonance imaging of the heart. At operation, a silver-whitish, smooth surfaced cystic mass was found attached to the free wall of the right atrium between the superior vena cava and the right atrial appendage with a broad base. Microscopically, the wall of the cyst was composed of stellate mesenchymal cells embedded within a myxoid matrix which was proved by alcian blue stain. To our knowledge, this type of cardiac myxoma has not been previously reported.

Expanding the histologic spectrum of mucinous tubular and spindle cell carcinoma of the kidney.

PubMed

Fine, Samson W; Argani, Pedram; DeMarzo, Angelo M; Delahunt, Brett; Sebo, Thomas J; Reuter, Victor E; Epstein, Jonathan I

2006-12-01

Mucinous tubular and spindle cell carcinomas (MTSCs) are polymorphic neoplasms characterized by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by pale mucinous stroma. Nonclassic morphologic variants and features of MTSC have not been well studied. We identified 17 previously unreported MTSCs from Surgical Pathology and consultative files of the authors and their respective institutions and studied their morphologic features. A total of 10/17 cases were considered "classic," as described above, with 5/10 showing at least focal (20% to 50%) tubular predominance without apparent mucinous matrix. Alcian blue staining revealed abundant (>50%) mucin in all classic cases. Seven of 17 MTSCs were classified as "mucin-poor," with little to no extracellular mucin appreciable by hematoxylin and eosin. Four of these cases showed equal tubular and spindled morphology, 2 cases showed spindle cell predominance (70%; 95%), and 1 case showed tubular predominance (90%). In 5/7 mucin-poor cases, staining for Alcian blue revealed scant (<10%) mucin in cellular areas with the other 2 cases having 30% mucin. Unusual histologic features identified in the 17 cases were: foamy macrophages (n=8), papillations/well formed papillae (n=6/n=1), focal clear cells in tubules (n=3), necrosis (n=3), oncocytic tubules (n=2; 40%, 5%), numerous small vacuoles (n=2), heterotopic bone (n=1), psammomatous calcification (n=1), and nodular growth with lymphocytic cuffing (n=1). An exceptional case contained a well-circumscribed, HMB45-positive angiomyolipoma within the MTSC. MTSCs may be "mucin-poor" and show a marked predominance of either of its principal morphologic components, which coupled with the presence of other unusual features such as clear cells, papillations, foamy macrophages, and necrosis, may mimic other forms of renal cell carcinoma. Pathologists must be aware of the spectrum of histologic findings within MTSCs to ensure their accurate diagnosis.

Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage.

PubMed

Ansari, Sahar; Diniz, Ivana M; Chen, Chider; Aghaloo, Tara; Wu, Benjamin M; Shi, Songtao; Moshaverinia, Alireza

2017-09-15

Cartilage tissue regeneration often presents a challenging clinical situation. Recently, it has been shown that Periodontal Ligament Stem Cells (PDLSCs) possess high chondrogenic differentiation capacity. In this study, we developed a stem cell delivery system based on alginate/hyaluronic acid (HA) loaded with TGF-β1 ligand, encapsulating PDLSCs; and investigated the chondrogenic differentiation of encapsulated cells in alginate/HA hydrogel microspheres in vitro and in vivo. The results showed that PDLSCs, as well as human bone marrow mesenchymal stem cells (hBMMSCs), as the positive control, were stained positive for both toluidine blue and alcian blue staining, while exhibiting high levels of gene expression related to chondrogenesis (Col II, Aggrecan and Sox-9), as assessed via qPCR. The quantitative PCR analyses exhibited that the chondrogenic differentiation of encapsulated MSCs can be regulated by the modulus of elasticity of hydrogel delivery system, confirming the vital role of the microenvironment, and the presence of inductive signals for viability and differentiation of MSCs. In vivo, histological and immunofluorescence staining for chondrogenic specific protein markers confirmed ectopic cartilage-like tissue regeneration inside transplanted hydrogels. PDLSCs presented significantly greater capability for chondrogenic differentiation than hBMMSCs (P < 0.05). Altogether, our findings confirmed that alginate/HA hydrogels encapsulating PDLSCs are a promising candidate for cartilage regeneration.

Zygomatic gland adenoma in a dog: histochemical and immunohistochemical evaluation.

PubMed

Giudice, Chiara; Marco, Rondena; Mirko, Radice; Luca, Mertel; Giorgio, Cammarata

2005-01-01

Orbital epithelial tumors in dogs are rare and most frequently malignant. Distinguishing their origin from the lacrimal or zygomatic gland is often challenging and is based mostly on tumor location. A case of adenoma involving the orbit in a 13-year-old, female, standard Schnauzer is reported. Histologically, the neoplasm was characterized by nests and cords of epithelial cells mostly forming small glandular structures. The origin of the tumor from the zygomatic gland was determined by histochemical characteristics (alcian blue pH 1 positive staining) of a small remnant of normal gland included within the tumor capsule. The benign nature of our finding was confirmed by follow-up information: 2 years after complete surgical removal of the mass no tumor recurrence or metastases was recorded.

Constructing bio-layer of heparin and type IV collagen on titanium surface for improving its endothelialization and blood compatibility.

PubMed

Zhang, Kun; Chen, Jun-ying; Qin, Wei; Li, Jing-an; Guan, Fang-xia; Huang, Nan

2016-04-01

The modification of cardiovascular stent surface for a better micro-environment has gradually changed to multi-molecule, multi-functional designation. In this study, heparin (Hep) and type IV collagen (IVCol) were used as the functional molecule to construct a bifunctional micro-environment of anticoagulation and promoting endothelialization on titanium (Ti). The surface characterization results (AFM, Alcian Blue 8GX Staining and fluorescence staining of IVCol) indicated that the bio-layer of Hep and IVCol were successfully fabricated on the Ti surface through electrostatic self-assembly. The APTT and platelet adhesion test demonstrated that the bionic layer possessed better blood compatibility compared with Ti surface. The adhesion, proliferation, migration and apoptosis tests of endothelial cells proved that the Hep/IVCol layer was able to enhance the endothelialization of the Ti surface. The in vivo animal implantation results manifested that the bionic surface could encourage new endothelialization. This work provides an important reference for the construction of multifunction micro-environment on the cardiovascular scaffold surface.

Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs

NASA Astrophysics Data System (ADS)

Sung, Baeckkyoung; Kim, Min Su; Lee, Byung-Cheon; Yoo, Jung Sun; Lee, Sang-Hee; Kim, Youn-Joong; Kim, Ki-Woo; Soh, Kwang-Sup

2008-02-01

There have been several reports on novel threadlike structures (NTSs) on the surfaces of the internal organs of rats and rabbits since their first observation by Bonghan Kim in 1963. To confirm this novel circulatory function, it is necessary to observe the flow of liquid through the NTS as well as the structurally corroborating channels in the NTS. In this article, we report on the measurement of the flow speed of Alcian blue solution in the NTSs on the organ surfaces of rabbits, and we present electron microscopic images depicting the cribrous cross-section with channels. The speed was measured as 0.3 ± 0.1 mm/s, and the flow distance was up to 12 cm. The flow was unidirectional, and the phase contrast microscopic images showed that the NTSs were strongly stained with Alcian blue. The ultrastructure of the NTSs revealed by cryo-scanning electron microscopy and high-voltage electron microscopy showed that (1) there were cell-like bodies and globular clumps of matter inside the sinus of the channel with thin strands of segregated zones which is a microscopic evidence of the liquid flow, (2) the sinuses have wall structures surrounded with extracellular matrices of collagenous fibers, and (3) there exists a cribriform structure of sinuses. To understand the mechanism for the circulation, a quantitative analysis of the flow speed has been undertaken applying a simplified windkessel model. In this analysis, it was shown that the liquid flow through the NTSs could be due to peristaltic motion of the NTS itself.

A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl.

PubMed

Demircan, Turan; İlhan, Ayşe Elif; Aytürk, Nilüfer; Yıldırım, Berna; Öztürk, Gürkan; Keskin, İlknur

2016-09-01

Axolotl (Ambystoma Mexicanum) has been emerging as a promising model in stem cell and regeneration researches due to its exceptional regenerative capacity. Although it represents lifelong lasting neoteny, induction to metamorphosis with thyroid hormones (THs) treatment advances the utilization of Axolotl in various studies. It has been reported that amphibians undergo anatomical and histological remodeling during metamorphosis and this transformation is crucial for adaptation to terrestrial conditions. However, there is no comprehensive histological investigation regarding the morphological alterations of Axolotl organs and tissues throughout the metamorphosis. Here, we reveal the histological differences or resemblances between the neotenic and metamorphic axolotl tissues. In order to examine structural features and cellular organization of Axolotl organs, we performed Hematoxylin & Eosin, Luxol-Fast blue, Masson's trichrome, Alcian blue, Orcein and Weigart's staining. Stained samples from brain, gallbladder, heart, intestine, liver, lung, muscle, skin, spleen, stomach, tail, tongue and vessel were analyzed under the light microscope. Our findings contribute to the validation of the link between newly acquired functions and structural changes of tissues and organs as observed in tail, skin, gallbladder and spleen. We believe that this descriptive work provides new insights for a better histological understanding of both neotenic and metamorphic Axolotl tissues. Copyright © 2016 Elsevier GmbH. All rights reserved.

Effect of vagal stimulation on gastric mucosal barrier in albino rats.

PubMed

Somasundaram, K; Ganguly, A K

1987-01-01

To study the influence of vagus nerves on gastric mucosal barrier in albino rats, gastric adherent mucus and mucosal epithelial neutral glycoproteins were quantitatively assessed after vagal stimulation at the cardio-esophageal region by a specially designed circular electrode. Gastric adherent mucus and epithelial mucus were studied from oxyntic and pyloric gland areas by Alcian blue binding and periodic acid Schiff's (PAS) staining method respectively. The results when compared with sham operated control animals showed increase in the visible mucus concurrent with decrease in PAS positive materials. The stimulation at the cardio-esophageal region of vagotomized animals did not produce these effects. This study indicates that in an acute condition, increased vagal influence is important in increasing mucus secretion and strengthening the first line of defence of the mucosal barrier.

Myxoma of the penis in an African pygmy hedgehog (Atelerix albiventris)

PubMed Central

TAKAMI, Yoshinori; YASUDA, Namie; UNE, Yumi

2016-01-01

A penile tumor (4 × 2.5 × 1 cm) was surgically removed from an African pygmy hedgehog (Atelerix albiventris) aged 3 years and 5 months. The tumor was continuous with the dorsal fascia of the penile head. Histopathologically, tumor cells were pleomorphic (oval-, short spindle- and star-shaped cells) with low cell density. Abundant edematous stroma was weakly positive for Alcian blue staining and positive for colloidal iron reaction. Tumor cells displayed no cellular atypia or karyokinesis. Tumor cell cytoplasm was positive for vimentin antibody, while cytoplasm and nuclei were positive for S-100 protein antibody. Tumor cell ultrastructure matched that of fibroblasts, and the rough endoplasmic reticulum was enlarged. The tumor was diagnosed as myxoma. This represents the first report of myxoma in a hedgehog. PMID:27784859

Myxoma of the penis in an African pygmy hedgehog (Atelerix albiventris).

PubMed

Takami, Yoshinori; Yasuda, Namie; Une, Yumi

2017-01-20

A penile tumor (4 × 2.5 × 1 cm) was surgically removed from an African pygmy hedgehog (Atelerix albiventris) aged 3 years and 5 months. The tumor was continuous with the dorsal fascia of the penile head. Histopathologically, tumor cells were pleomorphic (oval-, short spindle- and star-shaped cells) with low cell density. Abundant edematous stroma was weakly positive for Alcian blue staining and positive for colloidal iron reaction. Tumor cells displayed no cellular atypia or karyokinesis. Tumor cell cytoplasm was positive for vimentin antibody, while cytoplasm and nuclei were positive for S-100 protein antibody. Tumor cell ultrastructure matched that of fibroblasts, and the rough endoplasmic reticulum was enlarged. The tumor was diagnosed as myxoma. This represents the first report of myxoma in a hedgehog.

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

PubMed Central

1979-01-01

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity. PMID:512629

Gastric mucosal defence mechanism during stress of pyloric obstruction in albino rats.

PubMed

Somasundaram, K; Ganguly, A K

1987-04-01

1. The integrity of the gastric mucosa and its ability to secrete mucus are believed to be essential for protection of gastric mucosa against ulceration induced by aggressive factors active in any stress situation. This study involves a three-compartmental analysis of gastric mucosal barrier in pylorus-ligated albino rats. 2. Quantitative analyses of histologically identifiable gastric mucosal epithelial neutral glycoproteins and gastric adherent mucus from oxyntic and pyloric gland areas, and components of non-dialysable mucosubstances in gastric secretion were made under stress of pyloric obstruction for 4, 8, and 16 h durations. Epithelial mucin was identified by periodic acid-Schiff (PAS) staining technique and assessed from the ratio of gastric mucosal thickness to the depth of PAS positive materials in it. The remaining visible mucus adhered to the gastric mucosa was estimated by Alcian blue binding technique. The results were compared with that of identical control groups. 3. A significant reduction in mucosal epithelial PAS positive materials after 8 or 16 h of pylorus ligation was observed. 4. The Alcian blue binding capacity of the pyloric gland area was increased significantly after 4 h of pylorus ligation, while after 8 or 16 h it was reduced in both oxyntic and pyloric gland areas. 5. Significant reductions in the rate of gastric secretion and volume, as well as concentration of the components of non-dialysable mucosubstances, were observed, indicating decreased synthesis of mucus glycoproteins. 6. Disruption of the mucosal barrier may have occurred due to decreased mucus synthesis and acid-pepsin accumulation; both could be due to stress associated with gastric distension. 7. The present findings confirm the role of mucus in protecting the underlying gastric epithelium during stress. The adherent mucus offers a first line of defence and epithelial mucus a second line of defence.

Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

PubMed

Martínez, J; Martorell, J; Abarca, M L; Olvera, A; Ramis, A; Woods, L; Cheville, N; Juan-Sallés, C; Moya, A; Riera, A; Soto, S

2012-01-01

Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 μm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification. Copyright © 2011. Published by Elsevier Ltd.

Myxoid atypical fibroxanthoma: a previously undescribed variant.

PubMed

Patton, A; Page, R; Googe, P B; King, R

2009-11-01

Atypical fibroxanthomas (AFX) are dermal-based cutaneous tumors typically found in sun-damaged skin of the elderly. Histologic variants include spindle cell, clear cell, osteoid, osteoclastic, chondroid, pigmented, and granular cell. To date, myxoid change in AFX, has not been described. Four cases were retrieved from the consultation and surgical pathology files of Knoxville Dermatopathology Laboratory, Knoxville, Tennessee during a 4-year period. The clinical and histologic findings were reviewed and Alcian blue/periodic acid-Schiff (PAS) stain and panel of immunohistochemical stains was obtained. All 4 lesions occurred as solitary lesions in elderly males on the head and neck (2 cases) and upper extremity (2 cases). Histologically all tumors demonstrated a well-circumscribed, cellular lesion centered in the dermis and composed of a mix of atypical pleomorphic and spindle cells in a prominent myxomatous background. A junctional component was absent and the tumors did not arise from the epidermis or adnexal structures. Subcutaneous involvement was absent in all cases. Tumor cells were negative for melanocytic and epithelial markers. Positive staining was noted with CD10 (3/4 cases) and vimentin (4/4 cases). Myxoid change in AFX is rare and previously undescribed in the English literature. Myxoid change may be a prominent finding in benign and malignant cutaneous tumors and awareness of this variant of AFX will avoid misdiagnosis.

Effects of tretinoin pretreatment on TCA chemical peel in guinea pig skin.

PubMed Central

Kim, I. H.; Kim, H. K.; Kye, Y. C.

1996-01-01

This study was done to characterize the structural changes in the tretinoin pretreatment on trichloroacetic acid(TCA) chemical peel. In guinea pigs, the right halves pretreated with tretinoin and the left halves treated nothing were compared in their structural changes after TCA chemical peel. Epidermal thickness in the tretinoin pretreated group was almost the same in the first and second week. But epidermis of the TCA group increased continuously. In the first week, mitotic figures in the epidermis were more increased in the TCA group, but those in hair follicles were more increased in the tretinoin pretreated group. In the second week, mitotic figures in the epidermis were almost same in both group, but in hair follicles of the tretinoin pretreated group, mitotic figures were much more increased. In alcian blue staining, glycosaminoglycan was stained much more strongly in dermis of the TCA group in first week, but was more strongly stained in the tretinoin pretreated group in second week. On electron microscopic findings, the fibroblasts in upper dermis were larger and had plentier cytoplasm with more organelles in the tretinoin pretreated group. Conclusively, tretinoin pretreatment on TCA chemical peel sustained the effects of TCA longer and showed synergistic effects of TCA and induced enhanced wound healing. PMID:8878803

Relationship of gastric Helicobacter pylori infection to Barrett’s esophagus and gastro-esophageal reflux disease in Chinese

PubMed Central

Zhang, Jun; Chen, Xiao-Li; Wang, Kang-Min; Guo, Xiao-Dan; Zuo, Ai-Li; Gong, Jun

2004-01-01

AIM: To evaluate the relationship of Helicobacter pylori infection to reflux esophagitis (RE), Barrett’s esophagus (BE) and gastric intestinal metaplasia (IM). METHODS: RE, BE and gastric IM were determined by upper endoscopy. Patients were divided into 2 groups; those with squamocolumnar junction (SCJ) beyond gastroesophageal junction (GEJ) ≥ 3 cm (group A), and those with SCJ beyond GEJ < 3 cm (group B). Biopsy specimens were obtained endoscopically from just below the SCJ, gastric antrum along the greater and lesser curvature. Pathological changes and H pylori infection were determined by HE staining, Alcian blue staining and Giemsa staining. RESULTS: The prevalence of H pylori infection was 46.93%. There was no difference in the prevalence between males and females. The prevalence of H pylori infection decreased stepwise significantly from RE grade I to III. There was no difference in the prevalence between the two groups, and between long-segment and short-segment BE. In distal stomach, prevalence of H pylori infection was significantly higher in patients with IM than those without IM. CONCLUSION: There is a protective role of H pylori infection to GERD. There may be no relationship between H pylori infection of stomach and BE. H pylori infection is associated with the development of IM in the distal stomach. PMID:14991936

Ocular melanoma and mammary mucinous carcinoma in an African lion.

PubMed

Cagnini, Didier Q; Salgado, Breno S; Linardi, Juliana L; Grandi, Fabrizio; Rocha, Rafael M; Rocha, Noeme S; Teixeira, Carlos R; Del Piero, Fabio; Sequeira, Julio L

2012-09-25

Reports of neoplasms in Panthera species are increasing, but they are still an uncommon cause of disease and death in captive wild felids. The presence of two or more primary tumor in large felids is rarely reported, and there are no documented cases of ocular melanoma and mammary mucinous carcinoma in African lions. An ocular melanoma and a mammary mucinous carcinoma are described in an African lion (Panthera leo). The first tumour was histologically characterized by the presence of epithelioid and fusiform melanocytes, while the latter was composed of mucus-producing cells with an epithelial phenotype that contained periodic acid-Schiff (PAS) and Alcian blue staining mucins. Metastases of both tumor were identified in various organs and indirect immunohistochemistry was used to characterize them. Peribiliary cysts were observed in the liver. This is the first description of these tumor in African lions.

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

PubMed

González-Robles, Arturo; Castañón, Guadalupe; Cristóbal-Ramos, Ana Ruth; Hernández-Ramírez, Verónica Ivonne; Omaña-Molina, Maritza; Martínez-Palomo, Adolfo

2007-12-01

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

Histological features of the vomeronasal organ in the giraffe, Giraffa camelopardalis.

PubMed

Kondoh, Daisuke; Nakamura, Kentaro G; Ono, Yurie S; Yuhara, Kazutoshi; Bando, Gen; Watanabe, Kenichi; Horiuchi, Noriyuki; Kobayashi, Yoshiyasu; Sasaki, Motoki; Kitamura, Nobuo

2017-06-01

The vomeronasal organ (VNO) that preferentially detects species-specific substances is diverse among animal species, and its morphological properties seem to reflect the ecological features of animals. This histological study of two female reticulated giraffes (Giraffa camelopardalis reticulata) found that the VNO is developed in giraffes. The lateral and medial regions of the vomeronasal lumen were covered with sensory and nonsensory epithelia, respectively. The vomeronasal glands were positive for periodic acid-Schiff and alcian blue (pH 2.5) stains. The VNO comprises several large veins like others in the order Cetartiodactyla, suggesting that these veins function in a pumping mechanism in this order. In addition, numerous thin-walled vessels located immediately beneath the epithelia covering the lumen entirely surrounded the vomeronasal lumen. This sponge-like structure might function as a specific secondary pump in giraffes. © 2017 Wiley Periodicals, Inc.

Geraniol attenuates 4NQO-induced tongue carcinogenesis through downregulating the activation of NF-κB in rats.

PubMed

Madankumar, Arumugam; Tamilarasi, Sasivarnam; Premkumar, Thandavamoorthy; Gopikrishnan, Mani; Nagabhishek, Natesh; Devaki, Thiruvengadam

2017-10-01

Geraniol, an acyclic monoterpene found in lemon grass and aromatic herb oil, has been shown to exert antitumor and antioxidant activities against various cancer types. The objective of this study was to investigate the potential chemoprotective role of geraniol against 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis in male Wistar rats and furthermore to study anti-inflammatory mechanisms of action through possible NF-κB signaling. 4NQO was administered to rats at the dose of 50 ppm through drinking water to induce tongue cancer in 20 weeks. 4NQO provoked inflammation by upregulating the expressions of the p65 subunit nuclear factor kappa-β (NF-κB) in the nucleus, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Additionally, staining for immature and mature mast cells in cancer niche by toluidine blue staining and alcian blue-safranin staining showed more accumulation. Co-treatment of geraniol 200 mg/kg b.w. showed a significant decrease in the level of p65 NF-κB in the nucleus, and this might be due to the inhibition of NF-κB activation/translocation into nucleus, which was further confirmed by decreased immature and mature mast cell density and the expression of inflammatory downstream mediators such as TNF-α, IL-1β, COX-2, and iNOS. Collectively, our results suggested that geraniol as a potential anti-inflammatory agent having the capability to obstruct 4NQO initiated NF-κB activation and modulated the expression of inflammatory mediators.

The Use of Ancillary Stains in the Diagnosis of Barrett Esophagus and Barrett Esophagus-associated Dysplasia: Recommendations From the Rodger C. Haggitt Gastrointestinal Pathology Society.

PubMed

Srivastava, Amitabh; Appelman, Henry; Goldsmith, Jeffrey D; Davison, Jon M; Hart, John; Krasinskas, Alyssa M

2017-05-01

Barrett esophagus (BE) is a known risk factor for the development of esophageal adenocarcinoma. Pathologists play a critical role in confirming the diagnosis of BE and BE-associated dysplasia. As these diagnoses are not always straightforward on routine hematoxylin and eosin-stained slides, numerous ancillary stains have been used in an attempt to help pathologists confirm the diagnosis. On the basis of an in-depth review of the literature, the Rodger C. Haggitt Gastrointestinal Pathology Society provides recommendations regarding the use of ancillary stains in the diagnosis of BE and BE-associated dysplasia. Because goblet cells are almost always identifiable on routine hematoxylin and eosin-stained sections, there is insufficient evidence to justify reflexive use of Alcian blue (at pH 2.5) and/or periodic-acid Schiff stains on all esophageal biopsies to diagnose BE. In addition, the use of mucin glycoprotein immunostains and markers of intestinal phenotype (CDX2, Das-1, villin, Hep Par 1, and SOX9) are not indicated to aid in the diagnosis of BE at this time. A diagnosis of dysplasia in BE remains a morphologic diagnosis, and hence, ancillary stains are not recommended for diagnosing dysplasia. Although p53 is a promising marker for identifying high-risk BE patients, it is not recommended for routine use at present; additional studies are needed to address questions regarding case selection, interpretation, integration with morphologic diagnosis, and impact on clinical outcome. We hope that this review and our recommendations will provide helpful information to pathologists, gastroenterologists, and others involved in the evaluation of patients with BE and BE-associated dysplasia.

[A study on pathology and immunoglobulin of orbital tissues from cases with Grave's ophthalmopathy].

PubMed

Zhou, X; Luo, Q; Xia, R

1999-07-01

To investigate pathological changes and the expression, localization of immunoglobulins (IgA & IgE) in orbital tissues from patients with Grave's ophthalmopathy. Orbital tissues were obtained at surgery from 19 patients with Grave's ophthalmopathy and 17 control subjects. Immunohistochemical studies were carried out by using anti-IgA, anti-IgE antibody and a streptavidin-biotin-peroxidase detection system. In addition, hematoxylin-eosin, alcian blue and Masson trichrome stains were employed to demonstrate the pathologic changes in the tissues. 12 of 17 samples from patients exhibited IgA positive staining for the connective tissue associated with the extraocular muscles and orbicularis oculi, however only 2 control muscles reacted minimally. IgE positive material was found in association with the majority of leukocytes and with muscle fibers in 13 patients, but only 4 control subjects displayed mild reactions. Significant expression of IgE was more often demonstrated in patients with eye disease of short course than in those of longer course. These results support the notion that the eye muscle fiber and fibroblast are important targets of the orbital autoimmune reaction in Graves' ophthalmopathy in which IgA and IgE both play important roles.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Use of a novel monoclonal antibody in diagnosis of Barrett's esophagus.

PubMed

Griffel, L H; Amenta, P S; Das, K M

2000-01-01

A novel monoclonal antibody (MAbDAS-1), that specifically reacts with colonic but not small intestinal epithelium, recognizes specialized columnar epithelium (SCE) in the esophagus. The frequency of its reactivity in biopsy specimens of patients with endoscopically suspected Barrett's Esophagus (BE) is examined. Fifty-two biopsy specimens of the distal esophagus from 38 patients were tested by immunoperoxidase method using MAbDAS-1. Fifty-four samples of cardia-type mucosa biopsied from the stomach were used as controls. Results were compared with histology and Alcian blue/high iron diamine (AB/HID). Of the 52 specimens, 29 had glandular epithelium and the rest had only squamous epithelium. Ten were diagnosed to have SCE by histology. All 10 samples reacted with MAbDAS-1 and with Alcian blue. Of the remaining 19 specimens, five also reacted with MAbDAS-1. None of the squamous epithelium and cardia specimens reacted with MAbDAS-1. MAbDAS-1 may detect intestinal metaplasia of the esophagus of colonic phenotype in the absence of histological evidence of SCE.

Systematic significance of midrib vascular bundles in some Schefflera Spreng (Araliaceae) species

NASA Astrophysics Data System (ADS)

Noor-Syaheera, M. Y.; Noraini, T.; Aida-Shafreena, A. P.

2014-09-01

Anatomy study was undertaken on midrib vascular bundles of six Schefflera Spreng species, namely as S. obovatilimba, S. borneensis, S.kinabaluensis, S.lineamentorum, S. opacus and S.petiolosa. The genus Schefflera belongs to the family Araliaceae. The objective of this study is to determine variations in the midrib anatomical characteristics that can be used to identify species. Leaves samples were collected from various forest reserves in Sabah and Sarawak, Malaysia., were fixed in AA (Acetic acid: Alcohol, in a ratio of 1:3), the midrib parts then were sectioned using sliding microtome, were stained in Safranin and Alcian blue, been mounted in Eupharal and were observed under light microscope. Findings in this study have shown that all species have complex structure of vascular bundles. Each species has identical arrangement of vascular bundles and can be very useful for species identification. As a conclusion, variation in the midrib anatomical characteristics is outstanding and can has taxonomic value in the genus Schefflera respectively.

[Clinical problems in medical mycology: Problem number 51].

PubMed

Romero, Mercedes; Messina, Fernando; Marín, Emmanuel; Arechavala, Alicia; Negroni, Ricardo; Depardo, Roxana; Walker, Laura; Benchetrit, Andrés; Santiso, Gabriela

A 48 year-old immunocompetent woman, who had a nodular lesion in the neck and a dense infiltrate at the lower lobe of the left lung, presented at the Mycology Unit of Muñiz Hospital of Buenos Aires City. The pulmonary infiltrate disappeared spontaneously 3 months later. The histopathological study of the nodular lesion showed capsulated yeasts (mucicarmin and alcian blue positive stains) compatible with Cryptococcus. The mycological study of a new sample, obtained by a nodular puncture, allowed the isolation of yeasts, identified as Cryptococcus gattii (VGII). Latex test for Cryptococcus capsular antigen in serum was positive (1/100). CSF cultures rendered negative results. Fluconazole at a daily dose of 800mg was given during 45 days with partial improvement; as cultures from a new clinical sample were positive for Cryptococcus, the antimycotic was changed to itraconazole 400mg/day for 5 months, with an excellent clinical response. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Silicon nitride grids are compatible with correlative negative staining electron microscopy and tip-enhanced Raman spectroscopy for use in the detection of micro-organisms.

PubMed

Lausch, V; Hermann, P; Laue, M; Bannert, N

2014-06-01

Successive application of negative staining transmission electron microscopy (TEM) and tip-enhanced Raman spectroscopy (TERS) is a new correlative approach that could be used to rapidly and specifically detect and identify single pathogens including bioterrorism-relevant viruses in complex samples. Our objective is to evaluate the TERS-compatibility of commonly used electron microscopy (EM) grids (sample supports), chemicals and negative staining techniques and, if required, to devise appropriate alternatives. While phosphortungstic acid (PTA) is suitable as a heavy metal stain, uranyl acetate, paraformaldehyde in HEPES buffer and alcian blue are unsuitable due to their relatively high Raman scattering. Moreover, the low thermal stability of the carbon-coated pioloform film on copper grids (pioloform grids) negates their utilization. The silicon in the cantilever of the silver-coated atomic force microscope tip used to record TERS spectra suggested that Si-based grids might be employed as alternatives. From all evaluated Si-based TEM grids, the silicon nitride (SiN) grid was found to be best suited, with almost no background Raman signals in the relevant spectral range, a low surface roughness and good particle adhesion properties that could be further improved by glow discharge. Charged SiN grids have excellent particle adhesion properties. The use of these grids in combination with PTA for contrast in the TEM is suitable for subsequent analysis by TERS. The study reports fundamental modifications and optimizations of the negative staining EM method that allows a combination with near-field Raman spectroscopy to acquire a spectroscopic signature from nanoscale biological structures. This should facilitate a more precise diagnosis of single viral particles and other micro-organisms previously localized and visualized in the TEM. © 2014 The Society for Applied Microbiology.

Application of alternative fixatives to formalin in diagnostic pathology

PubMed Central

Gatta, L. Benerini; Cadei, M.; Balzarini, P.; Castriciano, S.; Paroni, R.; Verzeletti, A.; Cortellini, V.; De Ferrari, F.; Grigolato, P.

2012-01-01

Fixation is a critical step in the preparation of tissues for histopathology. The aim of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine stainings), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved to be the best fixative for morphology. The morphology obtained with Bouin was comparable to the one with formalin. Hollande was the best fixative for histochemistry. Bouin proved to be equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved the possibility to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis. PMID:22688293

Functional anatomy of the lacrimal gland in African black ostrich Struthio camelus domesticus in the embryonic and postnatal period.

PubMed

Klećkowska-Nawrot, Joanna; Goździewska-Harłajczuk, Karolina; Nowaczyk, Renata; Krasucki, Krzysztof

2015-03-25

The aim of the present study was morphological and histochemical analysis of the lacrimalgland (LG) in African black ostrich Struthio camelus domesticus in the embryonic and postnatalperiod. Studies were conducted on 50 ostriches aged between the 28th day of incubation until7 months old. Tissue sections were stained with haematoxylin and eosin, Azan trichrome,periodic acid-Schiff, Alcian blue pH 2.5, aldehyde fuchsin and Hale's dialysed iron. The LGin ostrich was classified as a tubulo-acinar type. The primordia of the lobes were determinedin the LG structure on the 28th day of incubation, whilst the weakly visible lobes with aciniand tubules were observed on the 40th day of incubation. Morphometric studies of the LGshowed steady growth, characterised by an increase in both length and width. Histometricmeasurements of lobe size showed little difference between the first, second and third agegroups, whilst in the fourth age group a marked increase in size of lobes was observed.The study showed that, apart from morphological changes, during the growth of the LGthe character of acid mucopolysaccharides changed. Sulphated acid mucopolysaccharideswere indicated, particularly with aldehyde fuchsin (AF) staining in the fourth age group.The Hale's dialysed iron (HDI) staining showed a low concentration of carboxylated acidmucopolysaccharides in the first and second age groups and a higher concentration in thethird and fourth age groups. Periodic acid-Schiff staining (PAS)-positive cells were observedin each age group, but only a small number of cells with a weakly PAS-positive reaction weredemonstrated in the first age group.

Cephalometric assessment of human fetal head specimens.

PubMed

Radlanski, R J; Heikinheimo, K; Gruda, A

2013-07-01

Past investigations of prenatal craniofacial growth have largely relied on histological sections. Few studies have taken measurements on three-dimensional representations (3D reconstruction, 3D CT, postmortem) or varying depth levels (ultrasound), and we know of no craniofacial growth studies done on cleared-and-stained specimens of whole fetal heads. This study comprised 14 human fetal head specimens cleared and stained with alizarin red and alcian blue. They had been stored in glycerol and represented weeks 8-12 of gestation, with crown-rump lengths ranging from 23-145 mm. These specimens were cephalometrically analyzed in norma frontalis and norma lateralis, which notably included the opportunity for side-to-side comparison. As the cranial membrane bones progressively approached each other, the orbits, maxilla, and mandible gradually grew wider. Likewise, the sagittal dimensions of the maxilla and mandible increased continuously and synchronically. We noted side-to-side differences ranging from 2-5 mm. Another notable finding concerned the inclination of the maxilla relative to the cranial base, which increased more on the right than on the left side. This is the first investigation presenting side-to-side comparative measurements of human fetal head specimens. Such measurements are essential in the quest toward validating the findings of other imaging techniques such as CT or MRI and-most importantly-intrauterine sonography.

Programmable Mechanobioreactor for Exploration of the Effects of Periodic Vibratory Stimulus on Mesenchymal Stem Cell Differentiation

PubMed Central

Cashion, Avery T.; Caballero, Montserrat; Halevi, Alexandra; Pappa, Andrew; Dennis, Robert G.

2014-01-01

Abstract A programmable bioreactor using a voice-coil actuator was developed to enable research on the effects of periodic vibratory stimulus on human and porcine mesenchymal stem cells (MSCs). We hypothesized that low frequency vibrations would result in a cartilage phenotype and higher frequency vibrations would result in a bone phenotype. The mechanical stimulation protocol is adjusted from a computer external to the incubator via a USB cable. Once programmed, the embedded microprocessor and sensor system on the bioreactor execute the protocol independent of the computer. In each test, a sinusoidal stimulus was applied to a culture plate in 1-min intervals with a 15-min rest following each, for a total of 15 h per day for 10 days. Frequencies of 1 and 100 Hz were applied to cultures of both human and porcine umbilical cord–derived MSCs. Chondrogenesis was determined by Alcian blue staining for glycosaminoglycans and an increased differentiation index (ratio of mRNA for collagen II and collagen I). Osteogenic differentiation was indicated with Alizarin red for calcium staining and increased bone morphogenetic protein 2 mRNA. One-hertz stimulation resulted in a cartilage phenotype for both human and porcine MSCs, while 100-Hz stimulation resulted in a bone phenotype. PMID:24570842

Biological Response of Osteoblastic and Chondrogenic Cells to Graphene-Containing PCL/Bioactive Glass Bilayered Scaffolds for Osteochondral Tissue Engineering Applications.

PubMed

Deliormanlı, Aylin M; Atmaca, Harika

2018-05-25

Graphene-containing 13-93 bioactive glass and poly(ε-caprolactone)-based bilayer, electrically conductive scaffolds were prepared for osteochondral tissue repair. Biological response of osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells to the composite scaffolds was assessed under mono-culture and co-culture conditions. Cytotoxicity was investigated using MTT assay, cartilage matrix production was evaluated by Alcian blue staining, and mineralization of both types of cells in the different culture systems was observed by Alizarin red S staining. Results showed that osteoblastic and chondrogenic cells utilized in the study did not show toxic response to the prepared scaffolds under mono-culture conditions and higher cell viability rates were obtained in co-culture conditions. Larger mineralized areas were determined under co-culture conditions and calcium deposition amount significantly increased compared with that in control group samples after 21 days. Additionally, the amount of glycosaminoglycans synthesized in co-culture was higher compared to mono-culture conditions. Electric stimulation applied under mono-culture conditions suppressed the viability of MC3T3-E1 cells whereas it enhanced the viability rates of ATDC5 cells. The study suggests that the designed bilayered osteochondral constructs have the potential for osteochondral defect repair.

Dermal arteritis of the nasal philtrum in a Giant Schnauzer and three Saint Bernard dogs.

PubMed

Torres, Sheila M F; Brien, Timothy O; Scott, Danny W

2002-10-01

Arteritis of the nasal philtrum is described in four dogs. Two of the Saint Bernards were related. The lesions were solitary, well-circumscribed, linear ulcers that were neither pruritic nor painful. The age of the dogs at the time the owners first noticed the lesion ranged from 3 to 6 years. The ulcers had been present for 0.5-5 years before diagnosis was pursued. Three of the dogs experienced repeated, mild episodes of arterial bleeding from the ulcers. Two dogs also experienced a severe episode of bleeding that required surgical intervention. Histopathological findings included a V-shaped ulcer, neutrophilic dermal inflammation subjacent to the ulcer and lymphoplasmacytic dermatitis bordering the ulcer. The most remarkable pathological findings were present in the deep dermal arteries and arterioles subjacent to the ulcer. The changes were characterized by subendothelial spindle cell proliferation with marked extracellular matrix deposition that stained blue with Alcian Blue (mucin) and Masson's trichrome (collagen) and resulted in intimal thickening, and stenosis of dermal arteries and arterioles. Immunohistochemical studies suggested that the proliferating spindle cells were of either myofibroblast or smooth muscle origin (actin and vimentin positive). Anti-inflammatory therapy (glucocorticoids; tetracycline and niacinamide; fish oil) may be beneficial for long-term control of this condition, however, long-term maintenance treatment appears to be necessary.

Frequency of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from the Douro River estuary, Portugal.

PubMed

Carrola, João; Fontaínhas-Fernandes, António; Pires, Maria João; Rocha, Eduardo

2014-02-01

Liver lesions in wild fish have been associated with xenobiotic exposure. Facing reports of pollution in the Douro River estuary (north of Portugal), we have been making field surveys using fishes and targeting histopathological biomarkers of exposure and effect. Herein, we intended to better characterize and report the rate of one poorly understood lesion-hepatocellular fibrillar inclusions (HFI)-found in European flounder (Platichthys flesus). With this report, we aimed to establish sound baseline data that could be viewed as a starting point for future biomonitoring, while offering the world's second only pool of field data on such a liver toxicopathic lesion, which could be compared with data available from the UK estuaries. Sampling was done in the Douro River estuary over 1 year. A total of 72 animals were fished with nets, in spring-summer (SS) and autumn-winter (AW) campaigns. Livers were processed for histopathology and both routine and special staining procedures (alcian blue, periodic acid Schiff (PAS), tetrazonium coupling reaction). Immunohistochemistry targeted AE1/AE3 (pan cytokeratins). The severity of the HFI extent was graded using a system with four levels, varying from 0 (absence of HFI) to 3 (high relative density of cells with HFI). Cells (isolated/groups) with HFI appeared in 35 % or more of the fish, in the total samples of each season, and over 40 % in more homogeneous sub-samples. There were no significant differences when comparing samples versus sub-samples or SS versus AW. When merging the data sets from the two seasons, the frequency of fish with HFI was ≈36 % for the total sample and ≈49 % for the sub-sample. The extreme group (biggest and smallest fish) revealed a HFI frequency of only 16 %, which differed significantly from the total and sub-sampled groups. Immunostaining and PAS were negative for the HFI, and alcian blue could, at times, faintly stain the inclusions. These were positive with the tetrazonium reaction. We showed the presence of HFI in European flounder from the Douro River estuary, proving that they are essentially protein in nature, that no seasonal changes existed in the HFI frequency, and that it was rarer in the smallest and biggest fish groups. Within the ranges of weight/size of our total sample, we estimate that the frequency of HFI in the local flounder is ≈35 %. That rate stands as a baseline value for future assessments, namely for biomonitoring purposes targeting correlations with the estuary pollution status.

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.

PubMed

Lee, H G; Cowman, M K

1994-06-01

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Distribution and heterogeneity of mast cells in female reproductive tract and ovary on different days of the oestrus cycle in Angora goats.

PubMed

Karaca, T; Arikan, S; Kalender, H; Yoruk, M

2008-08-01

The physiological distribution of mast cells (MCs) in the reproductive tract and ovary of 12 Angora goats was determined using light microscopic histochemical techniques. Uterus (corpus uteri and cornu uteri), uterine cervix, uterine tubes (isthmus and ampulla) and ovary samples were obtained by laparatomy from groups of animals during metoestrus, dioestrus and proestrus (days 5, 10 and 16 of the oestrous cycle). Tissues were fixed in Mota's fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 1% aqueous solution at pH 1.0 for 5 min and alcian blue-Safranin at pH 1.0 for 30 min. MCs were generally associated with blood vessels in all reproductive organs. In the uterus, they were concentrated mainly in the close of the uterine gland and deep stroma in the endometrium. Higher MC numbers were observed by toluidine blue staining in the uterus, uterine cervix and uterine tubes on days 10 (corpus uterine: 4.7 +/- 3.8 and cornu uterine: 4.9 +/- 3.5) and 16 (corpus uterine: 5.9 +/- 4.5 and cornu uterine: 5.4 +/- 2.4) of the oestrous cycle compared with day 5 (p < 0.05). Mast cells were not observed in the follicles, the corpus luteum and the underside of the surface epithelium of the ovarian cortex, but were observed in the interstitial cortical stroma and the ovarian medulla. In the ovary, MC numbers were significantly higher on day 16 of the oestrous cycle (cortex: 3.4 +/- 2.4 and medulla: 5.7 +/- 4.5, p < 0.05). Safranin-positive connective tissue MCs were not observed in the uterine tube on any occasion. These results indicate oestrous cycle-related changes in the number and location of MCs in goat reproductive organs.

Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair.

PubMed

Li, Yueying; Liu, Tie; Van Halm-Lutterodt, Nicholas; Chen, JiaYu; Su, Qingjun; Hai, Yong

2016-02-17

An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair. We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression. hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells. This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

Cell therapy: a therapeutic alternative to treat focal cartilage lesions.

PubMed

Gimeno, M J; Maneiro, E; Rendal, E; Ramallal, M; Sanjurjo, L; Blanco, F J

2005-11-01

Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Synovial membranes (n = 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments(micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment.

Comparison of Alcian blue and total carbohydrate assays for quantitation of transparent exopolymer particles (TEP) in biofouling studies.

PubMed

Li, Xu; Skillman, Lucy; Li, Dan; Ela, Wendell P

2018-04-15

Transparent exopolymer particles (TEP) and their precursors are gel-like acidic polysaccharide particles. Both TEP precursors and TEP have been identified as causal factors in fouling of desalination and water treatment systems. For comparison between studies, it is important to accurately measure the amount and fouling capacity of both components. However, the accuracy and recovery of the currently used Alcian blue based TEP measurement of different surrogates and different size fractions are not well understood. In this study, we compared Alcian blue based TEP measurements with a total carbohydrate assay method. Three surrogates; xanthan gum, pectin and alginic acid; were evaluated at different salinities. Total carbohydrate concentrations of particulates (>0.4 μm) and their precursors (<0.4 μm, >10 kDa) varied depending on water salinity and method of recovery. As xanthan gum is the most frequently used surrogate in fouling studies, TEP concentration is expressed as xanthan gum equivalents (mg XG eq /L) in this study. At a salinity of 35 mg/L sea salt, total carbohydrate assays showed a much higher particulate TEP fraction for alginic acid (38%) compared to xanthan gum (9%) and pectin (12%). The concentrations of particulate TEP therefore may only represent ∼10% of the total mass; while precursor TEP represents ∼80% of the total TEP. This highlights the importance of reporting both particulate and precursor TEP for membrane biofouling studies. The calculated concentrations of TEP and their precursors in seawater samples are also highly dependent on type of surrogate and resulting calibration factor. A linear correlation between TEP recovery and calibration factor was demonstrated in this study for all three surrogates. The relative importance and accuracy of measurement method, particulate size, surrogate type, and recovery are described in detail in this study. Copyright © 2017. Published by Elsevier Ltd.

The distribution patterns of COMP and matrilin-3 in septal, alar and triangular cartilages of the human nose.

PubMed

Wiggenhauser, Paul Severin; Schwarz, Silke; Rotter, Nicole

2018-05-02

The biomechanical characteristics of septal cartilage depend strongly on the distinct extracellular matrix of cartilage tissue; therefore, it is essential that the components of this matrix are identified and understood. Cartilage oligomeric matrix protein (COMP) and matrilin-3 are localised in articular cartilage. This study was the first to examine all subtypes of mature human nasal cartilages (alar, triangular and septal) with specific attention to the distribution of COMP and matrilin-3. Three whole fresh-frozen noses from human donors were dissected, and exemplary biopsies were examined using histochemical staining (haematoxylin and eosin and Alcian blue) and immunohistochemistry (collagen II, COMP and matrilin-3). The following three zones within the nasal cartilage were identified: superficial, intermediate and central. COMP was detected as highest in the intermediate zones in all three subtypes of nasal cartilage, whereas matrilin-3 was detected with pericellular deposition mainly within septal cartilage predominantly in the superficial zones. The distinct staining patterns of COMP and matrilin-3 underscore the different functional roles of both proteins in nasal cartilage. According to the literature, COMP might be involved with collagen II in the formation of networks, whereas matrilin-3 is reported to prevent ossification or regulate mechanosensitivity. The predominant staining observed in septal cartilage suggests matrilin-3's modulatory role because of its presence in the osteochondral junctional zone and given that the biomechanical load in septal cartilage is different from that in alar or triangular cartilage. In conclusion, COMP and matrilin-3 were detected in mature human nasal cartilage but displayed different staining patterns that might be explained by the functional roles of the respective matrix protein; however, further research is necessary to identify and define the functional aspects of this morphological difference.

Histological analysis of retraction pocket pars tensa of tympanic membrane in children.

PubMed

Urík, M; Hurník, P; Žiak, D; Machač, J; Šlapák, I; Motyka, O; Vaculová, J; Dvořáčková, J

2016-07-01

Histological and histochemical analysis of retraction pocket of pars tensa of tympanic membrane in children. Identification of morphological abnormalities in comparison with a healthy tympanic membrane as it is described in standard textbook. Identification of signs typical for cholesteatoma and support for a retraction theory of cholesteatoma formation. A prospective study analysing 31 samples of retraction pockets taken during surgery. University Hospital, Children's Medical Centre Samples of retraction pockets were processed by a standard process for light microscopy, stained by haematoxylin-eosin. Van Gieson's stain was used for differential staining of collagen, Verhoeff's stain for elastic fibre tissues, Alcian blue for acidic polysaccharides and PAS (Periodic Acid Schiff) method for basement membrane polysaccharides. The following findings were observed in the samples of retraction pockets: hyperkeratosis (100%), hypervascularisations (100%), subepithelial fragmented elastic fibres (96%), myxoid changes (87%), subepithelial inflammatory infiltration (84%), rete pegs (71%), papilomatosis (71%), intraepithelial inflammatory cellularizations, (48%), intraepithelial spongiosis (16%) and parakeratosis (3%). No basement membrane continuity interruptions were observed. Thickness of retraction pocket, thickness of epidermis, occurrence of rete pegs and frequency of fragmented elastic fibres was higher in a Grade III stage RP than Grade II stage RP (according to Charachon). Morphological abnormalities in the structure of retraction pockets in comparison with a healthy tympanic membrane were described. The changes are typical for a structure of cholesteatoma (these changes are common in matrix and perimatrix), supporting retraction theory of its origin. Our observations show that it is inflammation that probably plays a key role in the pathogenesis of retraction pocket. The frequency of some of the changes increases with the stage of retraction pocket (II-III according to Charachon). Basement membrane continuity interruptions are not typical for retraction pockets. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of extracellular matrix in chick paravertebral sympathetic ganglia.

PubMed

Luckenbill-Edds, L

1986-08-01

Alcian blue staining coupled with enzyme digestion or critical electrolyte staining revealed differences in the development of extracellular matrix (ECM) within sympathetic ganglia compared with the surrounding capsule. On day 5 of chick development (Hamburger-Hamilton stage 26) only hyaluronic acid (HA) could be detected in the ECM surrounding condensing primary ganglia. By day 7 (st 30) the ganglionic capsule contained HA, as well as sulfated glycosaminoglycans (GAGs), and this pattern continued into the adult stage. During the later stages of embryonic life (st 41-45) satellite cells appear, showing fine structural characteristics that point to their role in the secretion of intraganglionic ECM. Only during these stages could ECM be detected histochemically within ganglia, the same stages (days 15-19) when routine electron microscopic methods reveal collagen fibrils embedded in a granular ground substance. Thus, the intraganglionic environment appears as a separate compartment free of detectable amounts of GAG until late embryonic stages when ECM is secreted around satellite cells. This developmental pattern could represent a role of ECM in the histological stabilization of ganglia during the late stages of differentiation, since the appearance of intraganglionic ECM is correlated with the appearance of small dense-cored vesicles characteristic of adult neurons. The developmental pattern of ECM in differentiating sympathetic ganglia is compared with that of other tissues that undergo condensation and morphogenesis.

Age-related changes in the macula. A histopathological study of fifty Indian donor eyes.

PubMed

Biswas, Jyotirmay; Raman, Rajiv

2002-09-01

Age-related macular degeneration (ARMD) is clinically less common in India compared to the West. Therefore, clinicians are unfamiliar with histopathologic evidence of age-related macular changes in the Indian population. Fifty consecutive human donor eyes removed for corneal grafting were studied for gross, microscopic and histochemical features of age-related changes in the macula in the Indian population. A horizontal block was cut from the globe including the optic disc, and the macula. Six sections, 6 microns thick, were cut from three levels in the macula at a distance of 140 microns. These were stained with haemotoxylin-eosin, periodic acid-Schiff, Mallory, Masson trichrome, alcian blue and von Kossa stains. The presence of basal laminar deposits, drusen and thickening and calcification of Bruch's membrane in the macula were assessed at 400 x magnification using a modified version of Sark's classification. Twenty-four donor eyes (48%) had some form of age-related macular change. These included basal laminar deposits, hard drusen, soft drusen, extensive retinal pigment epithelium atrophy of the macula, and disciform degeneration of macula. A combination of changes was often seen. Age-related changes were more common in the seventh and eighth decade. Our study shows that histological changes characteristic of the early stages of age-related macular degeneration are fairly common in the Indian population. However, advanced macular changes are significantly rare.

Achilles tendinosis – a morphometrical study in a rat model

PubMed Central

Silva, Rafael Duarte; Glazebrook, Mark Anthony; Campos, Vinicius Castro; Vasconcelos, Anilton Cesar

2011-01-01

This study addresses the morphopathogenesis of Achilles tendinosis, using a rat model and presenting quantitative analysis of time-dependent histological changes. Thirty Wistar rats were used, randomly split in experimental and control groups. Animals of the experimental group were submitted to a treadmill running scheme. Five animals of each group were euthanized at four, eight and sixteen weeks. Achilles tendons were collected and processed routinely for histopath sections. Slides were stained by Hematoxylin-Eosin, Picrosirius Red, Alcian Blue, AgNOR, TUNEL and evaluated morphometrically. Cellular density decreased slightly along the time and was higher in the experimental group than in controls at fourth, eighth and sixteenth weeks. Fiber microtearing, percentual of reticular fibers and glycosaminoglycans content increased along the time and were higher in experimental group than in controls at all-time intervals. AgNOR labeling here interpreted as a marker of transcription activity was higher in the experimental groups than in controls at all-time intervals. Apoptotic cells were more frequent and diffusely distributed in tendinosis samples than in control groups. These results suggest that as mechanical overload is becoming chronic, cellular turnover and matrix deposition increases leading to tendinosis. The combination of staining techniques and morphometry used here to describe the evolution of lesions occurring in a rat model system has proved to be suited for the study of induced Achilles tendinosis. PMID:22076169

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Alginate Bead-Encapsulated PEDF Induces Ectopic Bone Formation In Vivo in the Absence of Co-Administered Mesenchymal Stem Cells.

PubMed

Elahy, Mina; Doschak, Michael R; Hughes, Jeffery D; Baindur-Hudson, Swati; Dass, Crispin R

2018-01-01

Bone defects can be severely debilitating and reduce quality of life. Osteoregeneration can alleviate some of the complications in bony defects. For therapeutic use in future, a single factor that can cause potent bone regeneration is highly preferred as it will be more costeffective, any off-target effects will be more easily monitored and potentially managed, and for ease of administration which would lead to better patient compliance and satisfaction. We demonstrate that pigment epithelium-derived factor (PEDF), one such factor that is known to be potent against angiogenesis, promotes osteoblastogenesis in mesenchymal stem cells in vitro, but does not need co-encapsulation of cells in alginate bead scaffolds for osteogeneration in vivo. Osteogenic differentiation by PEDF in vitro was confirmed with immunoblotting and immunocytochemical staining for bone markers (alkaline phosphatase, osteocalcin, osteopontin, collagen I), calcified mineral deposition, and assay for alkaline phosphatase activity. PEDF-mediated bone formation in a muscle pocket in vivo model was confirmed by microcomputed tomography (microCT), histology (haematoxylin and eosin, Alcian blue staining), immunostaining for bone markers and for collagen I-processing proteins (heat shock protein 47 and membrane type I matrix metalloproteinase). PEDF therefore presents itself as a promising biological for osteogeneration. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Three-Dimensional Scaffold from Decellularized Human Gingiva for Cell Cultures: Glycoconjugates and Cell Behavior

PubMed Central

Naderi, Somayeh; Khayat Zadeh, Jina; Mahdavi Shahri, Nasser; Nejad Shahrokh Abady, Khadijeh; Cheravi, Mojtaba; Baharara, Javad; Banihashem Rad, Seyed Ali; Bahrami, Ahmad Reza

2013-01-01

Objective: We studied both the presence of some carbohydrate compounds in a threedimensional (3D) matrix harvested from human gingiva and the cell behavior in this matrix. Materials and Methods: In this experimental research, in order to prepare 3D scaffolds, human palatal gingival biopsies were harvested and physically decellularized by freezethawing and sodium dodecyl sulfate (SDS). The scaffolds were placed within the rings of blastema tissues obtained from a pinna rabbit, in vitro. We evaluated the presence of glycoconjugatesand cellular behavior according to histological, histochemical and spectrophotometry techniques at one, two and three weeks after culture. One-way analysis of variance (ANOVA)comparedthe groups. Results: Extracellular matrix (ECM) remained after decellularization of tissue with 1% SDS. Glycoconjugate contents decreased meaningfully at a higher SDS concentration (p<0.0001). After culture of the ECM scaffold with blastema, we observed increased staining of alcian blue, periodic acid-Schiff (PAS) and toluidine blue in the scaffold and a number of other migrant cells which was caused by cell penetrationinto the scaffold. Spectrophotometry results showed an increase in glycosaminoglycans (GAGs) of the decellularized scaffolds at three weeks after culture. Conclusion: The present study has shown that a scaffold generated from palatal gingival tissue ECM is a suitable substrate for blastema cell migration and activity.This scaffold maypotentially be useful as a biological scaffold in tissue engineering applications. PMID:23862119

Influence of Layer-by-Layer Polyelectrolyte Deposition and EDC/NHS Activated Heparin Immobilization onto Silk Fibroin Fabric

PubMed Central

Elahi, M. Fazley; Guan, Guoping; Wang, Lu; King, Martin W.

2014-01-01

To enhance the hemocompatibility of silk fibroin fabric as biomedical material, polyelectrolytes architectures have been assembled through the layer-by-layer (LbL) technique on silk fibroin fabric (SFF). In particular, 1.5 and 2.5 bilayer of oppositely charged polyelectrolytes were assembled onto SFF using poly(allylamine hydrochloride) (PAH) as polycationic polymer and poly(acrylic acid) (PAA) as polyanionic polymer with PAH topmost. Low molecular weight heparin (LMWH) activated with 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was then immobilized on its surface. Alcian Blue staining, toluidine blue assay and X-ray photoelectron spectroscopy (XPS) confirmed the presence of heparin on modified SFF surfaces. The surface morphology of the modified silk fibroin fabric surfaces was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), and obtained increased roughness. Negligible hemolytic effect and a higher concentration of free hemoglobin by a kinetic clotting time test ensured the improved biological performance of the modified fibroin fabric. Overall, the deposition of 2.5 bilayer was found effective in terms of biological and surface properties of the modified fibroin fabric compared to 1.5 bilayer self-assembly technique. Therefore, this novel approach to surface modification may demonstrate long term patency in future in vivo animal trials of small diameter silk fibroin vascular grafts. PMID:28788601

Histological observation of a gelatin sponge transplant loaded with bone marrow-derived mesenchymal stem cells combined with platelet-rich plasma in repairing an annulus defect

PubMed Central

Xu, Xiang; Hu, Jianzhong; Lu, Hongbin

2017-01-01

Objective To research the histological characteristics of a gelatin sponge transplant loaded with goat BMSCs (bone marrow-derived mesenchymal stem cells) combined with PRP (platelet-rich plasma) in repairing an annulus defect. Method BMSCs were separated from the iliac crest of goats, sub-cultured and identified after the third generation. Then, PRP was obtained using blood from the jugular vein of goats via two degrees of centrifugation. In the animal experiments, the goats were divided into the following three groups: a sham group, an injury group and a therapeutic group. In the sham group, we decompressed the lamina and exposed the annulus fibrosus. In the injury group, we exposed the annulus fibrosus after decompression of the lamina and created a 1 × 1 cm defect in the annulus using surgical instruments. In the therapeutic group, after decompression of the lamina, we exposed the annulus, created a 1 × 1 cm defect using surgical instruments, and placed a gelatin sponge combined with BMSCs and PRP into the defect for a combined method of repair. Three, six and twelve weeks after the surgery, the previously damaged or undamaged annulus tissue was removed from the three groups. Then, the above tissue was assayed using HE (hematoxylin-eosin) staining, Masson trichrome staining, AB-PAS (Alcian blue-periodic acid Schiff) staining, and type II collagen staining and observed by microscopy. Results From the HE staining, we observed that the number of repair cells gradually increased. Compared to the injury group, the cell density and gross morphology of cells in the therapeutic group were closer to those of the sham group. As observed by Masson trichrome gelatin staining, many of the fibroblast cells or tissues were under repair, and as time progressed, the number of fibroblast cells and amount of tissue gradually increased. The results of the AB-PAS staining suggest that chondrocytes participated in the repair of the annulus. The level of type II collagen gradually increased, as determined by immunohistochemical staining. Conclusion Our results demonstrate that a gelatin sponge transplant loaded with BMSCs and PRP can effectively repair annulus defects. PMID:28178294

Effective isolation of primo vessels in lymph using sound- and ultrasonic-wave stimulation.

PubMed

Park, Do-Young; Lee, Hye-Rie; Rho, Min-Suk; Lee, Sang-Suk

2014-12-01

The effects of stimulation with sound and ultrasonic waves of a specific bandwidth on the microdissection of primo vessels in lymphatic vessels of rabbit were investigated. The primo vessels stained with alcian-blue dye injected in the lymph nodes were definitely visualized and more easily isolated by sound-wave vibration and ultrasonic stimulation applied to rabbits at various frequencies and intensities. With sound wave at 7 Hz and ultrasonic waves at 2 MHz, the probability of detecting the primo vessels was improved to 90%; however, without wave stimulation the probability of discovering primo vessels was about 50% only. Sound and ultrasonic waves at specific frequency bands should be effective for microdissection of the primo vessels in the abdominal lymph of rabbit. We suggest that oscillation of the primo vessels by sound and ultrasonic waves may be useful to visualize specific primo structure, and wave vibration can be a very supportive process for observation and isolation of the primo vessels of rabbits. Copyright © 2014. Published by Elsevier B.V.

A histochemical study of the distribution of lectin binding sites in the developing branchial area of the trout Salmo trutta.

PubMed Central

Rojo, M C; Blánquez, M J; González, M E

1996-01-01

A histochemical study of the branchial area of brown trout embryos from 35 to 71 d of incubation is reported. A battery of 6 different horseradish peroxidase-labelled lectins, the PAS reaction and Alcian blue staining were used to study the distribution of carbohydrate residues in glycoconjugates along the pharyngeal and branchial epithelia. Con A and WGA reacted at every site of the branchial region thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. WGA, DBA and SBA were good markers for the hatching gland cells (HGCs) and mucous cells. Other lectins, such as PNA and UEA I, reacted only for a short time at some sites during the considered period of incubation. From 35 d until posthatching stages, a manifest strong reaction was noted both in the dorsal epithelium of branchial arches and the HGCs as shown by SBA reactivity. This may be significant with regard to the controversial origin of HGCs, which is thought to be endodermal. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8982837

Anti-inflammatory effects of tilmicosin in a noninfectious mouse model of allergic asthma.

PubMed

Ci, Xinxin; Chu, Xiao; Xiang, Hua; Li, Xiangchao; Deng, Xuming

2011-12-01

Tilmicosin, a semi-synthetic tylosin-derived macrolide antibiotic commonly used by veterinarians, has been shown to possess anti-inflammatory activity. However, possible use in asthma treatment has not yet been studied. In this study, we investigated the anti-inflammatory properties of tilmicosin using a murine asthma model. BALB/c mice were sensitized and challenged by intraperitoneal (i.p.) or nasal administration of ovalbumin. Tilmicosin (10 and 20 mg/kg) treatment resulted in a marked reduction in the presence of several types of immune cells and cytokines in the bronchoalveolar lavage fluids of mice. Levels of ovalbumin-specific Immunoglobulin E (IgE) were significantly decreased following treatment with tilmicosin (10 and 20 mg/kg). Histological studies using H&E (haematoxylin and eosin) and AB-PAS (alcian blue-periodic acid-Schiff) staining demonstrated that tilmicosin substantially inhibited both ovalbumin-induced inflammatory cells in lung tissues and goblet cell hyperplasia in the airway. These findings provided new insight into the immunopharmacological role of tilmicosin in terms of its effects in a murine model of asthma.

Transitional metaplasia in intestinal epithelium of rats submitted to intestinal cystoplasty and treatment with L -lysine.

PubMed

Santos, Alessandra Marques Dos; Coelho, Joao Paulo Ferreira; Juanes, Camila de Carvalho; Azevedo, Rafael Barbosa de; Diniz, Clara Araujo; Jamacaru, Francisco Vagnaldo Fechine; Dornelas, Conceição Aparecida

2017-04-01

To evaluated the effects of L-lysine on the intestinal and urothelial epithelia in cystoplasty in rats. Twenty-eight 9-week-old rats were assigned to 4 groups: Group A (n=8) cystoplasty followed by administration of L-lysine (150 mg/kg body weight by gavage) for 30 weeks; Group B (n=8) cystoplasty + water for 30 weeks; Group C (n=6) L-lysine for 30 weeks; Group D (n=6) water for 30 weeks. On histopathology with hematoxylin and eosin, mild to moderate hyperplasia transitional was observed in at the site of anastomosis in all animals submitted to cystoplasty (Groups A and B), but "transitional metaplasia" of the intestinal glandular epithelium was more accentuated in Group A (p=0.045). No inflammatory cells, dysplasia or abnormalities were observed. Staining with Alcian blue revealed a substantial reduction of goblet cells and mucins in the colon segment (Groups A and B). The administration of L-lysine to rats accelerated the development of transitional metaplasia in the epithelium of the colon segment in cystoplasty.

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction.

PubMed

Chen, Chia-Chun; Liao, Cheng-Hao; Wang, Yao-Horng; Hsu, Yuan-Ming; Huang, Shih-Horng; Chang, Chih-Hung; Fang, Hsu-Wei

2012-03-01

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage-like structure with positive Alcian blue staining in the cartilage fragment-fibrin-MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. Copyright © 2011 Orthopaedic Research Society.

Immunohistochemical study of the digestive tract of Oligosarcus hepsetus

PubMed Central

Vieira-Lopes, Danielle A; Pinheiro, Nadja L; Sales, Armando; Ventura, Adriana; Araújo, Francisco G; Gomes, Iracema D; Nascimento, Aparecida A

2013-01-01

AIM: To describe the histology of the digestive tract and to investigate the occurrence of endocrine cells in Oligosarcus hepsetus (O. hepsetus). METHODS: The digestive tract (DT) of O. hepsetus was divided into esophagus, two stomach regions (glandular and non-glandular) and two intestinal regions (anterior and posterior). These specimens were processed by routine histological techniques and stained with hematoxylin-eosin, Gomori’s trichrome, periodic acid Schiff (PAS) and Alcian blue (AB). An immunohistochemical method using avidin-biotin-peroxidase was employed. RESULTS: The esophagus is lined with a non-keratinized stratified squamous epithelium that is reactive to PAS and AB. The stomach has a mucosa lined with a simple columnar epithelium with mucus-secreting cells that are reactive only to PAS. The intestine has a simple columnar epithelium with a brush border and goblet cells that are reactive to PAS and AB. Somatostatin, serotonin and cholecystokinin immunoreactive cells were identified throughout the DT. CONCLUSION: This study revealed adaptations for the species’ diet and showed that the distribution and relative frequency of immunoreactive cells are similar to those of other fish. PMID:23569337

Real time observation of mouse fetal skeleton using a high resolution X-ray synchrotron

PubMed Central

Chang, Dong Woo; Kim, Bora; Shin, Jae Hoon; Yun, Young Min; Je, Jung Ho; Hwu, Yeu kuang; Yoon, Jung Hee

2011-01-01

The X-ray synchrotron is quite different from conventional radiation sources. This technique may expand the capabilities of conventional radiology and be applied in novel manners for special cases. To evaluate the usefulness of X-ray synchrotron radiation systems for real time observations, mouse fetal skeleton development was monitored with a high resolution X-ray synchrotron. A non-monochromatized X-ray synchrotron (white beam, 5C1 beamline) was employed to observe the skeleton of mice under anesthesia at embryonic day (E)12, E14, E15, and E18. At the same time, conventional radiography and mammography were used to compare with X-ray synchrotron. After synchrotron radiation, each mouse was sacrificed and stained with Alizarin red S and Alcian blue to observe bony structures. Synchrotron radiation enabled us to view the mouse fetal skeleton beginning at gestation. Synchrotron radiation systems facilitate real time observations of the fetal skeleton with greater accuracy and magnification compared to mammography and conventional radiography. Our results show that X-ray synchrotron systems can be used to observe the fine structures of internal organs at high magnification. PMID:21586868

Dynamics of transparent exopolymer particle (TEP) production and aggregation during viral infection of the coccolithophore, Emiliania huxleyi.

PubMed

Nissimov, Jozef I; Vandzura, Rebecca; Johns, Christopher T; Natale, Frank; Haramaty, Liti; Bidle, Kay D

2018-06-19

Emiliania huxleyi produces calcium carbonate (CaCO 3 ) coccoliths and transparent exopolymer particles (TEP), sticky, acidic carbohydrates that facilitate aggregation. E. huxleyi's extensive oceanic blooms are often terminated by coccolithoviruses (EhVs) with the transport of cellular debris and associated particulate organic carbon (POC) to depth being facilitated by TEP-bound "marine snow" aggregates. The dynamics of TEP production and particle aggregation in response to EhV infection are poorly understood. Using flow cytometry, spectrophotometry, and FlowCam visualization of alcian blue (AB)-stained aggregates, we assessed TEP production and the size spectrum of aggregates for E. huxleyi possessing different degrees of calcification and cellular CaCO 3 :POC mass ratios, when challenged with two EhVs (EhV207 and EhV99B1). FlowCam imaging also qualitatively assessed the relative amount of AB-stainable TEP (i.e. blue:red ratio of each particle). We show significant increases in TEP during early phase EhV207-infection (∼24 hours) of calcifying strains and a shift towards large aggregates following EhV99B1-infection. We also observed the formation of large aggregates with low blue:red ratios, suggesting that other exopolymer substances contribute towards aggregation. Our findings show the potential for virus infection and the associated response of their hosts to impact carbon flux dynamics and provide incentive to explore these dynamics in natural populations. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Chondrogenesis of Human Adipose-Derived Stem Cells by In Vivo Co-graft with Auricular Chondrocytes from Microtia.

PubMed

Cai, Zhen; Pan, Bo; Jiang, Haiyue; Zhang, Lixia

2015-06-01

To evaluate the efficiency of chondrogenesis of human adipose-derived stem cells (ADSCs) induced by auricular chondrocytes from microtia via subcutaneous co-graft in nude mice. Human ADSCs and auricular chondrocytes were mixed at the ratio of 7:3 and suspended in 0.2 ml of Pluronic F-127 (5.0 × 10(7) cells/ml), and injected into Balb/c nude mice as the experimental group (Exp group). The same quantity of auricular chondrocytes (Ctr.1 group) or ADSCs (Ctr.2 group) in 0.2 ml of Pluronic F-127 was set as positive and negative control groups. The mixture of auricular chondrocytes (1.5 × 10(7) cells/ml) in 0.2 ml of Pluronic F-127 was set as the low concentration of chondrocyte control group (Ctr.3). At 8 weeks after grafting, the newly generated tissue pellets were isolated for morphological examination, haematoxylin and eosin staining, toluidine blue staining and safranin O staining of glycosaminoglycan (GAG), Masson's trichrome staining and immunohistochemical staining of type II collagen, and Verhoeff-iron-hematoxylin staining of elastic fibers. GAG content was determined by Alcian blue colorimetric method, and mRNA expression of type II collagen and aggrecan were examined by real-time PCR. Cartilage-like tissue with a white translucent appearance and good elasticity was generated in the Exp and Ctr.1 groups. The tissue pellets in the Ctr.2 and Ctr.3 groups were much smaller than those in the Ctr.1 group. The mature cartilage lacunas could be observed in the Exp and Ctr.1 groups, while were rarely seen in the Ctr.3 group and not observed in the Ctr.2 group. The expression of cartilage-specific extracellular matrix such as type II collagen, GAG content, aggrecan, and elastic fibers in the Exp group was similar to that in the Ctr.1 group, whereas the expression of these extracellular matrix substances was significantly lower in the Ctr.2 and Ctr.3 groups (both P < 0.01). Auricular chondrocytes from microtia can efficiently promote the chondrogenic differentiation and chondrogenesis of ADSCs by co-grafting in vivo. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Protocol for vital dye staining of corneal endothelial cells.

PubMed

Park, Sunju; Fong, Alan G; Cho, Hyung; Zhang, Cheng; Gritz, David C; Mian, Gibran; Herzlich, Alexandra A; Gore, Patrick; Morganti, Ashley; Chuck, Roy S

2012-12-01

To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Hyaluronan degrading silica nanoparticles for skin cancer therapy

NASA Astrophysics Data System (ADS)

Scodeller, P.; Catalano, P. N.; Salguero, N.; Duran, H.; Wolosiuk, A.; Soler-Illia, G. J. A. A.

2013-09-01

We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02787b

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

PubMed

Inaga, Sumire; Katsumoto, Tetsuo; Tanaka, Keiichi; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2007-04-01

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Prevalence of mabDAS-1 positivity in biopsy specimens from the esophagogastric junction.

PubMed

Rogge-Wolf, Claudia; Seldenrijk, Cornelis A; Das, Kiron M; Timmer, Robin; Breumelhof, Ronald; Smout, André J P M; Amenta, Peter S; Griffel, Louis H

2002-12-01

Intestinal metaplasia (IM) is a precursor for malignancies at the esophagogastric junction. A monoclonal antibody, mAbDAS-1, can probably identify cellular characteristics of IM before the appearance of goblet cells. The aim of this study was to examine the prevalence of mAbDAS-1 positivity in biopsies from the squamocolumnar junction (SCJ) and to correlate this positivity with the presence of IM and clinical findings. In 559 patients, reflux symptoms were scored, and the presence of reflux esophagitis and hiatus hernia was evaluated during endoscopy. Two biopsy specimens were obtained from the SCJ. In a subset of patients (n = 99), biopsies from the endoscopically defined cardiac region (2 cm distal to proximal margin of gastric folds) were available. Biopsy specimens were stained with hematoxylin and eosin, Alcian Blue, modified Giemsa, and mAbDAS-1. mAbDAS-1 positivity was observed in the SCJ biopsies of 201 of 486 (41.4%) patients without IM and in 64 of 73 (87.7%) patients with IM. Patients without IM but with antibody positivity showed similar histological characteristics as patients with IM at the SCJ. Biopsies of 123 of 559 patients (22%) revealed a columnar-cuboidal epithelium, which was found to be mAbDAS-1 positive in 64.2% (77 of 123). Tissue specimens from the cardiac region without IM stained positive in 14.2% (13 of 91), 12 of those also stained at the SCJ. In patients without IM, a high prevalence of mAbDAS-1 positivity was observed. Biopsies of these patients showed similar histological characteristics as patients with IM. Although not all patients exhibiting this reactivity may develop IM, mAbDAS-1 reactivity may help in the understanding of the histogenesis of IM at the SCJ.

Redifferentiation of dedifferentiated bovine articular chondrocytes enhanced by cyclic hydrostatic pressure under a gas-controlled system.

PubMed

Kawanishi, Makoto; Oura, Atsuhiro; Furukawa, Katsuko; Fukubayashi, Toru; Nakamura, Kozo; Tateishi, Tetsuya; Ushida, Takashi

2007-05-01

Hydrostatic pressure is one of the most frequently used mechanical stimuli in chondrocyte experiments. A variety of hydrostatic pressure loading devices have been used in cartilage cell experiments. However, no gas-controlled system with other than a low pressure load was used up to this time. Hence we used a polyolefin bag from which gas penetration was confirmed. Chondrocytes were extracted from bovine normal knee joint cartilage. After 3 passages, dedifferentiated chondrocytes were applied to form a pellet. These pellets were cultured in chemically defined serum-free medium with ITS+Premix for 3 days. Then 5 MPa of cyclic hydrostatic pressure was applied at 0.5 Hz for 4 h per day for 4 days. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 5-fold increase in the levels of aggrecan mRNA due to cyclic hydrostatic pressure load (p<0.01). Type II collagen mRNA levels were also upregulated 4-fold by a cyclic hydrostatic pressure load (p<0.01). Type I collagen mRNA levels were similarly reduced in the cyclic hydrostatic pressure load group and in the control group. The partial oxygen pressure (PO2) and partial carbon dioxide pressure (PCO2) of the medium in the bag reached equilibrium in 24 h, and no significant change was observed for 3 days afterwards. PO2 and PCO2 were very well controlled. The loaded pellet showed better safranin O/fast green staining than did the control pellet. Metachromatic staining by Alcian blue staining was found to be stronger in the loaded than in the control pellets. The extracellular matrices excretion of loaded pellets was higher than that of control pellets. These results suggest that gas-controlled cyclic hydrostatic pressure enhanced the cartilaginous matrix formation of dedifferentiated cells differentiated in vitro.

Simultaneous induction of Graves’ hyperthyroidism and Graves’ ophthalmopathy by TSHR genetic immunization in BALB/c mice

PubMed Central

Hu, Xiaohao; Song, Shiyu; Xu, Hui; Niu, Mengyuan; Wang, Hongwei; Wang, Jian

2017-01-01

Background Graves’ disease is the most common form of autoimmune thyroid disorder, characterized by hyperthyroidism due to circulating autoantibodies. To address the pathological features and establish a therapeutic approach of this disease, an animal model carrying the phenotype of Graves’ disease (GD) in concert with Graves’ Ophthalmopathy (GO) will be very important. However, there are no ideal animal models that are currently available. The aim of the present study is to establish an animal model of GD and GO disease, and its pathological features were further characterized. Methods A recombinant plasmid pcDNA3.1- T289 was constructed by inserting the TSHR A-subunit gene into the expression vector pcDNA3.1, and genetic immunization was successfully performed by intramuscular injection of the plasmid pcDNA3.1-T289 on female 8-week-old BALB/c mice. Each injection was immediately followed by in vivo electroporation using ECM830 square wave electroporator. Morphological changes of the eyes were examined using 7.0T MRI scanner. Levels of serum T4 and TSHR antibodies (TRAb) were assessed by ELISA. The pathological changes of the thyroid and orbital tissues were examined by histological staining such as H&E staining and Alcian blue staining. Results More than 90% of the immunized mice spontaneously developed goiter, and about 80% of the immunized mice manifested increased serum T4 and TRAb levels, combined with hypertrophy and hyperplasia of thyroid follicles. A significantly increased synthesis of hyaluronic acid was detected in in the immunized mice compared with the control groups. Conclusion We have successfully established an animal model manifesting Graves’ hyperthyroidism and ophthalmopathy, which provides a useful tool for future study of the pathological features and the development of novel therapies of the diseases. PMID:28319174

Meningoceles, meningomyeloceles, and encephaloceles: a neuro-dermatopathologic study of 132 cases.

PubMed

Berry, A D; Patterson, J W

1991-06-01

Because there have been few comprehensive histopathologic studies of meningomyeloceles and related malformations, we undertook a systematic study of these lesions. One hundred and thirty two cases were obtained from our surgical pathology files; these included 38 meningoceles, 71 meningomyeloceles, and 23 encephaloceles. Tissue sections were stained with hematoxylin and eosin; special stains included trichrome, alcian blue, Fontana-Masson, Nissl, Holzer, and immunoperoxidase for glial fibrillary acidic protein. Epithelial changes included ulceration, atrophy, or nevoid hyperplasia of the epidermis, and loss of appendages. Mesodermal features included fibrous zones resembling dura, subarachnoid tissue or scar (99% of cases), increased numbers of blood vessels (83%), hypertrophy of arrector pili muscle (42%), lipoma formation (38%), and immature skeletal muscle fibers (5%) that rarely intermingled with neuropil-like matrix. The latter tissue was identified in 71% of cases and included neurons, astrocytes, oligodendroglia, and ependyma. Forty-eight percent of cases included peripheral nerve fibers or roots, and some fibers formed onion bulb or Pacinian corpuscle-like structures. Meningothelial cells were observed in 26% of cases and sometimes formed recognizable whorls. Choroid plexus was noted in 3 cases, one example showing an unusual dystrophic calcification that formed long parallel spicules. Pigmented dendritic cells were observed within zones of fibrous tissue in 10% of cases. These malformations involve complex arrangements of cutaneous, neuroectodermal, and mesodermal elements. Because they may be encountered by dermatopathologists, familiarity with the microscopic features of dysraphic lesions is essential.

Ontogeny of the Appendicular Skeleton in Melanosuchus niger (Crocodylia: Alligatoridae).

PubMed

Vieira, Lucélia Gonçalves; Santos, André Luiz Quaqliatto; Lima, Fabiano Campos; Mendonça, Sônia Helena Santesso Teixeira de; Menezes, Lorena Tannus; Sebben, Antônio

2016-08-01

The objective of the present study was to analyze chondrogenesis and the ossification pattern of the limbs of Melanosuchus niger in order to contribute with possible discussions on homology and the fusion pattern of autopodial elements and phylogeny. In the Reserva Extrativista do Lago Cuniã, Rondônia, Brazil, six nests were marked and two eggs removed from each nest at 24-hour intervals until hatching. Embryos were cleared using KOH; bone tissue was stained with alizarin red S and cartilage with Alcian blue. Routine staining with HE was also performed. In the pectoral girdle, the scapula showed ossification centers before the coracoid process. In the pelvic girdle, the ilium and the ischium were condensed as a single cartilage, although ossification took place through two separate centers, forming distinct elements in the adult. The pubis developed from an independent cartilaginous center with free end, which reflects its function in breathing. In the initial stages, the stylopodium and the zeugopodium developed from the condensation of a Y-shaped cartilage in the limbs, and differentiation of the primary axis and digital arch were observed. The greatest changes were observed in the mesopodia. In their evolution, Crocodylia underwent a vast reduction in the number of autopodial elements as a consequence of fusions and ossification of some elements. This study shows that the chondrogenesis and ossification sequences are dissociated. Moreover, the differences between M. niger and other species show clear variation in the patterns for these events in Alligatoridae.

Ultrastructure and composition of Call-Exner bodies in bovine follicles.

PubMed

van Wezel, I L; Irving-Rodgers, H F; Sado, Y; Ninomiya, Y; Rodgers, R J

1999-05-01

Call-Exner bodies are present in ovarian follicles of a range of species including human and rabbit, and in a range of human ovarian tumors. We have also found structures resembling Call-Exner bodies in bovine preantral and small antral follicles. Hematoxylin and eosin staining of single sections of bovine ovaries has shown that 30% of preantral follicles with more than one layer of granulosa cells and 45% of small (less than 650 microns) antral follicles have at least one Call-Exner body composed of a spherical eosinophilic region surrounded by a rosette of granulosa cells. Alcian blue stains the spherical eosinophilic region of the Call-Exner bodies. Electron microscopy has demonstrated that some Call-Exner bodies contain large aggregates of convoluted basal lamina, whereas others also contain regions of unassembled basal-lamina-like material. Individual chains of the basal lamina components type IV collagen (alpha 1 to alpha 5) and laminin (alpha 1, beta 2 and delta 1) have been immunolocalized to Call-Exner bodies in sections of fresh-frozen ovaries. Bovine Call-Exner bodies are presumably analogous to Call-Exner bodies in other species but are predominantly found in preantral and small antral follicles, rather than large antral follicles. With follicular development, the basal laminae of Call-Exner bodies change in their apparent ratio of type IV collagen to laminin, similar to changes observed in the follicular basal lamina, suggesting that these structures have a common cellular origin.

Mucinous (colloid) adenocarcinomas secrete distinct O-acylated forms of sialomucins: a histochemical study of gastric, colorectal and breast adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Poveda, M A; Segura, D I

2001-12-01

Mucinous (colloid) adenocarcinomas represent a distinct group of tumours defined by the presence of large amounts of extracellular mucins. By using histochemical methods, we analysed mucins secreted by mucinous versus non-mucinous adenocarcinomas and looked for differential secretion profiles. Sixty-four adenocarcinomas were studied (23 colorectal, 17 gastric, and 24 breast tumours). Thirty-two tumours were of the colloid type. The following methods were applied to paraffin tissue sections: (i) Alcian blue (pH 2.5) and periodic acid-Schiff (PAS); (ii) high iron diamine and Alcian blue (pH 2.5); (iii) periodic acid borohydride, potassium hydroxide, and PAS; (iv) periodic acid-thionine Schiff, potassium hydroxide, and PAS; and (v) periodic acid-borohydride and PAS. Most adenocarcinomas secreted acidic mucins, with sialomucins predominating over sulfomucins, except for non-mucinous adenocarcinomas of the breast which showed predominant neutral mucins. All mucinous adenocarcinomas contained C9-O-acyl sialic acid as mono, di(C8,C9)-, or tri(C7,C8,C9)-O-acyl forms. Acidic mucins secreted by the majority of non-colloid adenocarcinomas consisted of non-O-acylated sialomucins. C9-O-acylation of sialic acid is a characteristic feature of mucinous adenocarcinomas and can be readily detected by histochemical methods.

The structure of the cutaneous pedal glands in the banded snail Cepaea hortensis (Müller, 1774).

PubMed

von Byern, Janek; Cyran, Norbert; Klepal, Waltraud; Rudoll, Livia; Suppan, Johannes; Greistorfer, Sophie

2018-02-01

Although gastropods have been crawling through the ocean and on the land for 60 million years, we still know very little about the sticky mucus produced in their foot. Most research has been focused on marine species in particular and, to a lesser extent, on the well-known terrestrial species Arion vulgaris and Cornu aspersum. Within this study, we aim to characterize the foot anatomy of a smaller representative of the family Helicidae, the banded snail Cepaea hortensis. We are particularly interested in the microanatomy of the foot glands, their position, and the histochemical nature of their secretory content. Characterization of the dorsal foot region of Cepaea hortensis reveals four glands, differing in their size and in the granules produced. Histochemically, three of them react positively for sugars (PAS staining and lectin affinity tests for mannose, glucose and N-acetyl-d-glucosamine) and acidic proteins (positive Alcian blue and Toluidine blue staining), indicating the presence of acidic glycosaminoglycans. The fourth gland type does not react to any of these dyes. The ventral pedal region includes two different gland types, which are positive for the presence of acidic glycoproteins, with a lectin affinity for mannose only. A comparison with Helix pomatia indicates differences regarding the number of glands and their contents. In Helix, only three gland types are described in the dorsal region of the foot, which show a similar granular appearance but nevertheless differ in their chemical composition. Congruently, there are two gland types in the ventral region in both species, whereas in Helix an additional sugar moiety is found. This raises the question whether these differences between the pedal glandular systems of both helicid species are the result of protection or size-related adaptations, as they occur in the same habitat. © 2017 Wiley Periodicals, Inc.

Corneal clouding in Alport syndrome.

PubMed

Herwig, Martina C; Eter, Nicole; Holz, Frank G; Loeffler, Karin U

2011-03-01

Alport syndrome is a hereditary basement membrane disease that typically involves the kidney, the cochlea, and the eyes. Characteristic ocular problems include posterior polymorphous corneal dystrophy, lenticonus, and dot-and-fleck retinopathy. A 48-year-old male patient with Alport syndrome presented with corneal and retinal changes. In 2003, he was diagnosed with posterior polymorphous corneal dystrophy and received a corneal transplant in his left eye in 2007 because of progressive deterioration in visual acuity. At this time, a lamellar macular hole was diagnosed in his right eye. The removed corneal button was examined by light and electron microscopy and by immunohistochemistry. Histology revealed not only endothelial changes but also a marked irregular thickening of the epithelial basement membrane and of Bowman layer. Alcian blue staining demonstrated an accumulation of mucopolysaccharides in the Bowman layer. The presented changes underline the great variation of ocular disorders related to Alport syndrome. To our knowledge, this is one of the first reports describing histologic corneal findings in Alport syndrome. Only a few cases with accumulation of mucopolysaccharides in the Bowman layer have been described previously, none of them being associated with Alport syndrome. Besides, anterior corneal alterations and corneal clouding seem to be uncommon in patients suffering from Alport syndrome.

Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

PubMed

Zhou, Haibo; Shi, Jianmin; Zhang, Chao; Li, Pei

2018-02-28

Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis. © 2018 The Author(s).

Prickly Pear Cactus (Opuntia ficus indica var. saboten) Protects Against Stress-Induced Acute Gastric Lesions in Rats

PubMed Central

Kim, Seung Hyun; Jeon, Byung Ju; Kim, Dae Hyun; Kim, Tae Il; Lee, Hee Kyoung; Han, Dae Seob; Lee, Jong-Hwan; Kim, Tae Bum; Kim, Jung Wha

2012-01-01

Abstract The protective activity of prickly pear cactus (Opuntia ficus indica var. saboten) fruit juice and its main constituent, betanin, were evaluated against stress-induced acute gastric lesions in rats. After 6 h of water immersion restraint stress (WIRS), gastric mucosal lesions with bleeding were induced in Sprague–Dawley rats. Pretreatment of a lyophilized powder containing O. ficus indica var. saboten fruit juice and maltodextrin (OFSM) and betanin significantly reduced stress lesions (800–1600 mg/kg). Both OFSM and betanin effectively prevented the decrease in gastric mucus content as detected by alcian blue staining. In addition, OFSM significantly suppressed WIRS-induced increases in the level of gastric mucosal tumor necrosis factor-α and myeloperoxidase (MPO). Betanin alone was only effective in decreasing MPO. These results revealed the protective activity of OFSM against stress-induced acute gastric lesions and that betanin may contribute to OFSM's gastric protective activity, at least in part. When OFSM and betanin were taken together, OFSM exerted gastroprotective activity against stress-induced gastric lesions by maintaining gastric mucus, which might be related to the attenuation of MPO-mediated damage and proinflammatory cytokine production. PMID:23062184

Prickly pear cactus (Opuntia ficus indica var. saboten) protects against stress-induced acute gastric lesions in rats.

PubMed

Kim, Seung Hyun; Jeon, Byung Ju; Kim, Dae Hyun; Kim, Tae Il; Lee, Hee Kyoung; Han, Dae Seob; Lee, Jong-Hwan; Kim, Tae Bum; Kim, Jung Wha; Sung, Sang Hyun

2012-11-01

The protective activity of prickly pear cactus (Opuntia ficus indica var. saboten) fruit juice and its main constituent, betanin, were evaluated against stress-induced acute gastric lesions in rats. After 6 h of water immersion restraint stress (WIRS), gastric mucosal lesions with bleeding were induced in Sprague-Dawley rats. Pretreatment of a lyophilized powder containing O. ficus indica var. saboten fruit juice and maltodextrin (OFSM) and betanin significantly reduced stress lesions (800-1600 mg/kg). Both OFSM and betanin effectively prevented the decrease in gastric mucus content as detected by alcian blue staining. In addition, OFSM significantly suppressed WIRS-induced increases in the level of gastric mucosal tumor necrosis factor-α and myeloperoxidase (MPO). Betanin alone was only effective in decreasing MPO. These results revealed the protective activity of OFSM against stress-induced acute gastric lesions and that betanin may contribute to OFSM's gastric protective activity, at least in part. When OFSM and betanin were taken together, OFSM exerted gastroprotective activity against stress-induced gastric lesions by maintaining gastric mucus, which might be related to the attenuation of MPO-mediated damage and proinflammatory cytokine production.

Characterization of midrib vascular bundles of selected medicinal species in Rubiaceae

NASA Astrophysics Data System (ADS)

Nurul-Syahirah, M.; Noraini, T.; Latiff, A.

2016-11-01

An anatomical study was carried out on mature leaves of five selected medicinal species of Rubiaceae from Peninsular Malaysia. The chosen medicinal species were Aidia densiflora, Aidia racemosa, Chasallia chartacea, Hedyotis auricularia and Ixora grandifolia. The objective of this study is to determine the taxonomic value of midrib anatomical characteristics. Leaves samples were collected from Taman Paku Pakis, Universiti Kebangsaan Malaysia, Bangi, Selangor and Kledang Saiong Forest Reserve, Perak, Malaysia. Leaves samples then were fixed in spirit and acetic acid (3:1), the midrib parts then were sectioned using sliding microtome, cleared using Clorox, stained in Safranin and Alcian blue, mounted in Euparal and were observed under light microscope. Findings in this study have shown all species have collateral bundles. The midrib vascular bundles characteristics that can be used as tool to differentiate between species or genus are vascular bundles system (opened or closed), shape and arrangement of main vascular bundles, presence of both additional and medullary vascular bundles, position of additional vascular bundles, shape of medullary vascular bundles, presence of sclerenchyma cells ensheathed the vascular bundles. As a conclusion, midrib anatomical characteristics can be used to identify and discriminate medicinal plants species studied in the Rubiaceae.

Comparison of methylene blue/gentian violet stain to Gram's stain for the rapid diagnosis of gonococcal urethritis in men.

PubMed

Taylor, Stephanie N; DiCarlo, Richard P; Martin, David H

2011-11-01

We compared a simple, one-step staining procedure using a mixture of methylene blue and gentian violet to Gram stain for the detection of gonococcal urethritis. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97.3% and 99.6%, respectively. There was a 100% correlation between the 2 methods.

A Versatile Giemsa Protocol for Permanent Nuclear Staining of Fungi

Treesearch

A. Dan Wilson

1992-01-01

A variety of cytological stains and staining procedures including Giemsa-HCL, acetic-or-cein, propionic-carmine, iron haema-toxylin, safranin O, aniline blue or trypan blue, toluidine blue and basic fuchsin or Feulgen stain have been used to investigate the nuclear condition of reproductive and somatic structures of fungi. Fluorescent stains such as acriflavin acridine...

Rapid alkaline methylene blue supravital staining for assessment of anterior segment infections.

PubMed

Kiuchi, Katsuji

2016-01-01

To present the Löffler's alkaline methylene blue technique of staining eye discharges in eyes with anterior segment infections. The Löffler's alkaline methylene blue staining method is a simple staining technique that can be used to differentiate bacterial, viral, and fungal infections. It is a cationic dye that stains cells blue because the positively charged dye is attracted to negatively charged particles such as polyphosphates, DNAs, and RNAs. Specimens collected from patients by swabbing are smeared onto microscope slides and the methylene blue solution is dropped on the slide. The slide is covered with a glass cover slip and examined under a microscope. The entire time from the collection to the viewing is about 30 seconds. Histopathological images of the conjunctival epithelial cells and neutrophils in eye discharges were dyed blue and the nuclei were stained more intensely blue. Bacterial infections consisted mainly of neutrophils, and viral infections consisted mainly of lymphocytes. Löffler's alkaline methylene blue staining can be done in about 30 seconds for diagnosis. Even though this is a one color stain, it is possible to infer the cause of the infection by detection of the absence of bacteria and/or fungi in context of the differential distribution of neutrophils and lymphocytes.

Solid intraocular xanthogranuloma in three Miniature Schnauzer dogs.

PubMed

Zarfoss, Mitzi K; Dubielzig, Richard R

2007-01-01

Macrophages that contain abundant intracytoplasmic lipid are called 'foam cells'. In four canine globes submitted to the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), foam cells formed a solid intraocular mass. The purpose of this study was to describe the histopathologic findings in these cases. The electronic COPLOW database (1993-2006) was searched for the diagnosis of 'foam cell tumor'. Clinical history, gross pathology and histopathology (5-micron sections, hematoxylin and eosin and Alcian blue periodic acid Schiff) were reviewed in all cases. Cases were included if the globe was grossly filled by a solid mass and if all intraocular structures were effaced by lipid-laden foam cell macrophages admixed with birefringent, Alcian blue-positive crystals oriented in stellate patterns. All three patients (four globes) satisfying the selection criteria were Miniature Schnauzers. In all cases the clinical history included diabetes mellitus, hyperlipidemia and chronic bilateral uveitis that was interpreted to be lens-induced. All globes were enucleated because of glaucoma. The term solid intraocular xanthogranuloma was used to describe these cases because the intraocular contents were effaced by a solid mass of foam cells and birefringent crystals. The cases in this report suggest that diabetic Miniature Schnauzers with hyperlipidemia are at risk for lipid and macrophage-rich uveitis, which may in some cases form a solid inflammatory intraocular mass, precipitate glaucoma, and lead to enucleation.

Ontogeny of the larynx and flight ability in Jamaican fruit bats (Phyllostomidae) with considerations for the evolution of echolocation.

PubMed

Carter, Richard T; Adams, Rick A

2014-07-01

Echolocating bats have adaptations of the larynx such as hypertrophied intrinsic musculature and calcified or ossified cartilages to support sonar emission. We examined growth and development of the larynx relative to developing flight ability in Jamaican fruit bats to assess how changes in sonar production are coordinated with the onset of flight during ontogeny as a window for understanding the evolutionary relationships between these systems. In addition, we compare the extent of laryngeal calcification in an echolocating shrew species (Sorex vagrans) and the house mouse (Mus musculus), to assess what laryngeal chiropteran adaptations are associated with flight versus echolocation. Individuals were categorized into one of five developmental flight stages (flop, flutter, flap, flight, and adult) determined by drop-tests. Larynges were cleared and stained with alcian blue and alizarin red, or sectioned and stained with hematoxylin and eosin. Our results showed calcification of the cricoid cartilage in bats, represented during the flap stage and this increased significantly in individuals at the flight stage. Thyroid and arytenoid cartilages showed no evidence of calcification and neither cricoid nor thyroid showed significant increases in rate of growth relative to the larynx as a whole. The physiological cross-sectional area of the cricothyroid muscles increased significantly at the flap stage. Shrew larynges showed signs of calcification along the margins of the cricoid and thyroid cartilages, while the mouse larynx did not. These data suggest the larynx of echolocating bats becomes stronger and sturdier in tandem with flight development, indicating possible developmental integration between flight and echolocation. © 2014 Wiley Periodicals, Inc.

Gallbladder mucin production and calcium carbonate gallstones in children.

PubMed

Sayers, Craig; Wyatt, Judy; Soloway, Roger D; Taylor, Donald R; Stringer, Mark D

2007-03-01

In contrast to adults, calcium carbonate gallstones are relatively common in children. Their pathogenesis is poorly understood. Cystic duct obstruction promotes calcium carbonate formation in bile and increases gallbladder mucin production. We tested the hypothesis that mucin producing epithelial cells would be increased in gallbladders of children with calcium carbonate gallstones. Archival gallbladder specimens from 20 consecutive children who had undergone elective cholecystectomy for cholelithiasis were examined. In each case, gallstone composition was determined by Fourier transform infrared microspectroscopy. Gallbladder specimens from six children who had undergone cholecystectomy for conditions other than cholelithiasis during the same period were used as controls. Multiple sections were examined in a blinded fashion and scored semiquantitatively for mucin production using two stains (alcian blue and periodic acid-Schiff). Increased mucin staining was observed in 50% or more epithelial cells in five gallbladder specimens from seven children with calcium carbonate stones, compared to 5 of 13 with other stone types (P = 0.17) and none of the control gallbladders (P = 0.02). Gallbladders containing calcium carbonate stones were significantly more likely than those containing other stone types or controls to contain epithelial cells with the greatest mucin content (P = 0.03). Gallbladders containing calcium carbonate stones were also more likely to show the ulcer-associated cell lineage. These results demonstrate an increase in mucin producing epithelial cells in gallbladders from children containing calcium carbonate stones. This supports the hypothesis that cystic duct obstruction leading to increased gallbladder mucin production may play a role in the development of calcium carbonate gallstones in children.

Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822. Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix.

PubMed

Vieira, Tuane C R G; Costa-Filho, Adilson; Salgado, Norma C; Allodi, Silvana; Valente, Ana-Paula; Nasciutti, Luiz E; Silva, Luiz-Claudio F

2004-02-01

Acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica, has a major disaccharide repeating unit of -->4)-2-acetyl,2-deoxy-alpha-d-glucopyranose(1-->4)-2-sulfo-alpha-l-idopyranosyluronic acid (1-->, making it structurally related to both heparin and heparan sulfate. It has been suggested that this glycosaminoglycan is polydisperse, with an average molecular mass of 29 kDa and known minor disaccharide sequence variants containing unsulfated iduronic acid. Acharan sulfate was found to be located in the body of this species using alcian blue staining and it was suggested to be the main constituent of the mucus. In the present work, we provide further information on the structure and compartmental distribution of acharan sulfate in the snail body. Different populations of acharan sulfate presenting charge and/or molecular mass heterogeneities were isolated from the whole body, as well as from mucus and from the organic shell matrix. A minor glycosaminoglycan fraction susceptible to degradation by nitrous acid was also purified from the snail body, suggesting the presence of N-sulfated glycosaminoglycan molecules. In addition, we demonstrate the in vivo metabolic labeling of acharan sulfate in the snail body after a meal supplemented with [35S]free sulfate. This simple approach might be applied to the study of acharan sulfate biosynthesis. Finally, we developed histochemical assays to localize acharan sulfate in the snail body by metachromatic staining and by histoautoradiography following metabolic radiolabeling with [35S]sulfate. Our results show that acharan sulfate is widely distributed among several organs.

The fake fat phenomenon in organizing pleuritis: a source of confusion with desmoplastic malignant mesotheliomas.

PubMed

Churg, Andrew; Cagle, Philip; Colby, Thomas V; Corson, Joseph M; Gibbs, Allen R; Hammar, Samuel; Ordonez, Nelson; Roggli, Victor L; Tazelaar, Henry D; Travis, William D; Wick, Mark

2011-12-01

We report 9 patients with pleural biopsies referred because of concern about infiltration of what appeared to be chest wall fat by pan-keratin-positive spindled cells, a finding that led to a consideration of desmoplastic mesothelioma. All patients showed pleural effusions/pleural thickening on computed tomographic scan. Pleural biopsy showed a greatly thickened and fibrotic paucicellular pleura with circular fat-like spaces and, sometimes, adjacent oblate spaces mostly deep in the fibrotic area. Indistinct, keratin-positive, spindle cells arranged parallel to the pleural surface coursed between these fat-like spaces. S-100 stains were negative around the fat-like spaces. Vimentin stains showed that the spaces did not have a cellular lining of any kind. Sometimes the spaces contained faintly hematoxyphilic material that was Alcian blue positive, and similar material was seen in the fibrotic stroma. Follow-up with periods ranging from 6 to 30 months revealed that 8 cases had stable disease on chest imaging or by clinical findings. One case had slowly progressive pleural thickening. These observations suggest that spaces resembling fat may be encountered in fibrotic pleurae and that horizontally oriented keratin-positive spindled cells between the fat-like spaces deep in the fibrotic portion of a thickened pleura represent a benign finding seen in some cases of organizing pleuritis/fibrothorax. The spaces themselves are probably artifacts derived from the biopsy procedure and/or cutting artifacts. In contrast, in true desmoplastic mesotheliomas there is downward, rather than horizontal, growth of keratin-positive spindled cells running between clearly definable fat cells.

Oocyte adhesiveness and embryonic development of Astyanax bimaculatus (Linnaeus, 1758) (Pisces: Characidae).

PubMed

Weber, André Alberto; Arantes, Fábio Pereira; Sato, Yoshimi; Rizzo, Elizete; Bazzoli, Nilo

2013-05-01

This study shows for the first time the presence of a jelly coat on oocytes of neotropical Characiformes fish. This structure could be responsible for the adhesiveness of Astyanax bimaculatus oocytes, a species widely distributed in South America including in the São Francisco River basin in Brazil. Adult specimens of A. bimaculatus were submitted to artificial reproduction in order to analyse the egg morphology and embryonic development. The eggs were fertilised and kept in incubators with a water temperature of 24°C so that embryogenesis could be monitored. Ovulated and unfertilised oocytes were also collected and submitted to routine histological techniques. Astyanax bimaculatus oocytes were found to be spherical, yellowish, and covered by a thin jelly coat with a slightly adhesive surface. The mean oocyte diameter was 1.03 ± 0.03 mm, the perivitelline space was 0.21 ± 0.02 mm and the jelly coat's thickness was 0.04 ± 0.01 mm. Positive periodic acid-Schiff (PAS) stain and Alcian blue stain pH 2.5 indicated the presence of neutral glycoproteins, and carboxylated acid glycoconjugates on the jelly coat that formed mucosubstances that may be associated with egg adhesiveness. At a water temperature of 24°C, blastopore closure and hatching occurred at 5 h and 17 h after fertilisation, respectively. The results of this study provide essential information for phylogenetic studies and for a better understanding of the reproductive strategy of A. bimaculatus, currently included in the incertae sedis group of the Characidae family due to the lack of monophyly among the families of the group.

Renaut bodies in nerves of the trunk of the African elephant, Loxodonta africana.

PubMed

Witter, Kirsti; Egger, Gunter F; Boeck, Peter

2007-05-01

Renaut bodies are loosely textured, cell-sparse structures in the subperineurial space of peripheral nerves, frequently found at sites of nerve entrapment. The trunk of the elephant is a mobile, richly innervated organ, which serves for food gathering, object grasping and as a tactile organ. These functions of the trunk lead to distortion and mechanical compression of its nerves, which can therefore be expected to contain numerous Renaut bodies. Samples of the trunk wall of an adult African elephant (Loxodonta africana) were examined histologically using conventional staining methods, immunohistochemistry, and lectin histochemistry. Architecture of nerve plexuses and occurrence of Renaut bodies in the elephant trunk were compared with those in tissues surrounding the nasal vestibule of the pig. Prominent nerve plexuses were found in all layers of the elephant trunk. Almost all (81%) nerve profiles contained Renaut bodies, a basophilic, discrete subperineurial layer resembling cushions around the nerve core. In contrast, Renaut bodies were seen in only 15% of nerve profiles in the porcine nasal vestibule. Within Renaut bodies, fusiform fibroblasts and round, ruff-like cells were placed into a matrix of acidic glycosaminoglycans with delicate collagen and very few reticular fibers. The turgor of this matrix is thought to protect nerves against compression and shearing strain. Renaut bodies are readily stained with alcian blue (pH 2.5) favorably in combination with immunohistochemical markers of nerve fibers. They should be regarded as a physiological response to repeated mechanical insults and are distinct from pathological alterations. alterations. (c) 2007 Wiley-Liss, Inc.

Search for optimized conditions for sealing and storage of bypass vessels: influence of preservation solution and filling pressure on the degree of endothelialization

PubMed Central

Weiss, Dominik Roger; Juchem, Gerd; Eblenkamp, Markus; Kemkes, Bernhard Michael; Gansera, Brigitte; Geier, Michael; Nees, Stephan

2010-01-01

The aim of the present study was to develop methods for the rapid assessment of intimal quality of coronary bypass segments of venous origin, and to prevent endothelial damage by improved intraoperative handling of graft segments. Particular attention was paid to the influence of the composition of the preservation solution and the intravasal filling pressure on the degree of endothelialization. Intrava-sal exposure to Alcian blue at pH<3 resulted in highly specific staining of intimal regions with functionally or structurally damaged endothelium. Standardization of preparation, staining and image acquisition of the intimal surface of graft remnants and subsequent computer-aided planimetry of these images made it possible for the first time to perform rapid serial investigations for quality control of bypass grafts. Using saline as the rinsing and intraoperative storage medium resulted in the loss of more than 50% of the endothelium at intravasal pressures of 0-100 mmHg. Increasing the pressure resulted eventually in complete de-endothelialization. In contrast, grafts incubated in a customized plasma derivative tolerated pressures of up to 200 mmHg with no significant endothelial loss; and even after exposure to 1,000 mmHg (10 times the average mean arterial pressure!) more than 70% of the endothelium were intact and vital. These findings imply strongly that the quality of aortocoronary bypass grafts of venous origin can be improved substantially by the use of a plasma derivative solution for intraoperative preservation and by monitoring and controlling the intravasal pressures reached during sealing and storage. PMID:20369036

Evidence of the gastroprotective and anti- Helicobacter pylori activities of β-mangostin isolated from Cratoxylum arborescens (vahl) blume

PubMed Central

Sidahmed, Heyam Mohamed Ali; Hashim, Najihah Mohd; Mohan, Syam; Abdelwahab, Siddig Ibrahim; Taha, Manal Mohamed Elhassan; Dehghan, Firouzeh; Yahayu, Maizatulakmal; Ee, Gwendoline Cheng Lian; Loke, Mun Fai; Vadivelu, Jamuna

2016-01-01

Purpose β-Mangostin (BM) from Cratoxylum arborescens demonstrated various pharmacological activities such as anticancer and anti-inflammatory. In this study, we aimed to investigate its antiulcer activity against ethanol ulcer model in rats. Materials and methods BM was isolated from C. arborescens. Gastric acid output, ulcer index, gross evaluation, mucus production, histological evaluation using hematoxylin and eosin and periodic acid–Schiff staining and immunohistochemical localization for heat shock protein 70 (HSP70) and Bax proteins were investigated. Possible involvement of reduced glutathione, lipid peroxidation, prostaglandin E2, antioxidant enzymes, superoxide dismutase and catalase enzymes, radical scavenging, nonprotein sulfhydryl compounds, and anti-Helicobacter pylori were investigated. Results BM showed antisecretory activity against the pylorus ligature model. The pretreatment with BM protect gastric mucosa from ethanol damaging effect as seen by the improved gross and histological appearance. BM significantly reduced the ulcer area formation, the submucosal edema, and the leukocytes infiltration compared to the ulcer control. The compound showed intense periodic acid–Schiff staining to the gastric mucus layer and marked amount of alcian blue binding to free gastric mucus. BM significantly increased the gastric homogenate content of prostaglandin E2 glutathione, superoxide dismutase, catalase, and nonprotein sulfhydryl compounds. The compound inhibited the lipid peroxidation revealed by the reduced gastric content of malondialdehyde. Moreover, BM upregulate HSP70 expression and downregulate Bax expression. Furthermore, the compound showed interesting anti-H. pylori activity. Conclusion Thus, it could be concluded that BM possesses gastroprotective activity, which could be attributed to the antisecretory, mucus production, antioxidant, HSP70, antiapoptotic, and anti-H. pylori mechanisms. PMID:26834460

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

PubMed

Werkmeister, J A; Adhikari, R; White, J F; Tebb, T A; Le, T P T; Taing, H C; Mayadunne, R; Gunatillake, P A; Danon, S J; Ramshaw, J A M

2010-09-01

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions. 2010. Published by Elsevier Ltd. All rights reserved.

Corneal edema and permanent blue discoloration of a silicone intraocular lens by methylene blue.

PubMed

Stevens, Scott; Werner, Liliana; Mamalis, Nick

2007-01-01

To report a silicone intraocular lens (IOL) stained blue by inadvertent intraoperative use of methylene blue instead of trypan blue and the results of experimental staining of various lens materials with different concentrations of the same dye. A "blue dye" was used to enhance visualization during capsulorhexis in a patient undergoing phacoemulsification with implantation of a three-piece silicone lens. Postoperatively, the patient presented with corneal edema and a discolored IOL. Various IOL materials were experimentally stained using methylene blue. Sixteen lenses (4 silicone, 4 hydrophobic acrylic, 4 hydrophilic acrylic, and 4 polymethylmethacrylate) were immersed in 0.5 mL of methylene blue at concentrations of 1%, 0.1%, 0.01%, and 0.001%. These lenses were grossly and microscopically evaluated for discoloration 6 and 24 hours after immersion. The corneal edema resolved within 1 month after the initial surgical procedure. After explantation, gross and microscopic analyses of the explanted silicone lens revealed that its surface and internal substance had been permanently stained blue. In the experimental study, all of the lenses except the polymethylmethacrylate lenses were permanently stained by methylene blue. The hydrophilic acrylic lenses showed the most intense blue staining in all dye concentrations. This is the first clinicopathological report of IOL discoloration due to intraocular use of methylene blue. This and other tissue dyes may be commonly found among surgical supplies in the operating room and due diligence is necessary to avoid mistaking these dyes for those commonly used during ocular surgery.

Multiplication free neural network for cancer stem cell detection in H-and-E stained liver images

NASA Astrophysics Data System (ADS)

Badawi, Diaa; Akhan, Ece; Mallah, Ma'en; Üner, Ayşegül; ćetin-Atalay, Rengül; ćetin, A. Enis

2017-05-01

Markers such as CD13 and CD133 have been used to identify Cancer Stem Cells (CSC) in various tissue images. It is highly likely that CSC nuclei appear as brown in CD13 stained liver tissue images. We observe that there is a high correlation between the ratio of brown to blue colored nuclei in CD13 images and the ratio between the dark blue to blue colored nuclei in H&E stained liver images. Therefore, we recommend that a pathologist observing many dark blue nuclei in an H&E stained tissue image may also order CD13 staining to estimate the CSC ratio. In this paper, we describe a computer vision method based on a neural network estimating the ratio of dark blue to blue colored nuclei in an H&E stained liver tissue image. The neural network structure is based on a multiplication free operator using only additions and sign operations. Experimental results are presented.

Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis.

PubMed

Qin, Zhan-Fen; Zhou, Jing-Ming; Cong, Lin; Xu, Xiao-Bai

2005-10-01

We examined potential ecotoxic effects of polychlorinated biphenyl (PCB)3, PCB5, Aroclor 1254, and Aroclor 1242 on Xenopus laevis. Tadpoles were exposed to PCBs from stage 46/47 (system of Nieuwkoop and Faber) to the completion of metamorphosis. We demonstrated, to our knowledge for the first time, forelimb malformations caused by PCBs (malformation rate, > 70%). The malformed forelimbs were fixed in the adduction-backward rotation position and could not move. Therefore, malformed male frogs were destined to have no offspring, because they could not grasp the females with their forelimbs to mate. Alcian blue-alizarin red double-staining indicated that the forelimb malformation resulted from the shoulder abnormality. Compared with the normal shoulder joint, the proximal humerus with the humerus inter-rotated 90 degrees in the abnormal shoulder joint. Moreover, testes from more than a third of male frogs with exposed to PCBs exhibited feminization to different degrees at gross morphology and histology, with fewer or abnormal spermatogonia and oocytes. Gonadal abnormalities would lead directly to reproductive dysfunction and population decline. These results suggest that PCBs have potentially ecotoxic effects on amphibian populations. We infer that PCBs could play roles in amphibian malformations and population declines, at least at sites that are polluted heavily with PCBs.

OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.

2016-03-01

The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.

An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

PubMed

Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

2015-11-01

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

PubMed Central

2012-01-01

Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material. PMID:22429795

Rebamipide increases mucin-like substance contents and periodic acid Schiff reagent-positive cells density in normal rabbits.

PubMed

Urashima, Hiroki; Takeji, Yasuhiro; Okamoto, Takashi; Fujisawa, Shigeki; Shinohara, Hisashi

2012-06-01

The effects of rebamipide on the number of periodic acid Schiff reagent (PAS)-positive cells in the conjunctiva, the mucin content in the cornea and conjunctiva of normal rabbits, and desiccation-induced corneal damage in vivo were examined. Rebamipide (0.1%-3%) was applied 6 times a day for 14 days, and the PAS-positive cell count in the bulbar conjunctiva was measured by impression cytology. The amount of conjunctival and corneal mucin-like substances was measured by Alcian blue binding. The corneal damage model was created by desiccation from air flow at room temperature. The level of corneal damage was determined by scoring the area stained with rose bengal and fluorescein dye. Rebamipide increased the number of PAS-positive cells in the conjunctiva when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances of the conjunctiva and cornea. Moreover, 1% rebamipide was also found to lower the rose bengal scores of the cornea in the corneal damage model by desiccation. Rebamipide is a possible candidate drug for treatment of cornea and conjunctival epithelial damage due to its mucin-like substance increasing action, for instance, in the treatment of dry eye disease.

Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides.

PubMed

Ercole, Claudia; Cacchio, Paola; Botta, Anna Lucia; Centi, Valeria; Lepidi, Aldo

2007-02-01

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25-40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

Effect of sex on histological and histochemical structures of different parts of the kidney in Japanese quail.

PubMed

Mobini, Behzad; Abdollahi, MohammadHossein

2016-09-01

The aim of the present study was to investigate the effect of gender on the histological and histochemical structures of different anatomical regions of the kidney in Japanese quail (Coturnix japonica). Tissue samples from cranial, middle and caudal divisions of each kidney were obtained from 20 male and 20 female adult, healthy Japanese quail. The sections stained with hematoxylin & eosin ( H & E: ), Masson's trichrome, Verhoeff's, Alcian blue (pH 2.5), Periodic acid-Schiff, and Gomori's method for reticulum. Unusual findings of the kidney in Japanese quail were the presence of three types of nephrons, all the connective tissue fibers in capsule and interlobular septa and AB-reactions of the proximal convoluted cells. No significant sex-based differences were found. The various histological structures of the kidney showed no significant differences among different divisions of the left and right kidneys. It is concluded that the general histological and histochemical properties of the kidney in Japanese quail were similar to those of chickens and some other species, but that there were also some differences. One of the major differences was brush border of interdigitating microvilli on luminal surface of collecting ducts in Japanese quail. © 2016 Poultry Science Association Inc.

Expression and distribution of hyaluronic acid and CD44 in unphonated human vocal fold mucosa.

PubMed

Sato, Kiminori; Umeno, Hirohito; Nakashima, Tadashi; Nonaka, Satoshi; Harabuchi, Yasuaki

2009-11-01

The tension caused by phonation (vocal fold vibration) is hypothesized to stimulate vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) to accelerate production of extracellular matrices. The distribution of hyaluronic acid (HA) and expression of CD44 (a cell surface receptor for HA) were examined in human vocal fold mucosae (VFMe) that had remained unphonated since birth. Five specimens of VFMe (3 adults, 2 children) that had remained unphonated since birth were investigated with Alcian blue staining, hyaluronidase digestion, and immunohistochemistry for CD44. The VFMe containing MFe were hypoplastic and rudimentary. The VFMe did not have a vocal ligament, Reinke's space, or a layered structure, and the lamina propria appeared as a uniform structure. In the children, HA was distributed in the VFMe containing MFe. In the adults, HA had decreased in the VFMe containing MFe. In both groups, the VFSCs in the MFe and the fibroblasts in the lamina propria expressed little CD44. This study supports the hypothesis that the tensions caused by vocal fold vibration stimulate the VFSCs in the MFe to accelerate production of extracellular matrices and form the layered structure. Phonation after birth is one of the important factors in the growth and development of the human VFMe.

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization.

PubMed

Mrázová, H; Koller, J; Kubišová, K; Fujeríková, G; Klincová, E; Babál, P

2016-06-01

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.

Extracellular accumulations in the avian pituitary gland: histochemical analysis in two species of Indian wild birds.

PubMed

Mohanty, Banalata

2006-01-01

Extracellular accumulations of two distinct types, colloid-filled follicles and fibrous-material-containing cysts, were observed in the pituitary gland of two species of Indian wild birds, Halcyon smyrnensis perpulchra and Lonchura striata striata. Colloid follicles were regular structures and distributed throughout the pars distalis (PD). The fibrous cysts were irregular structures, bigger than the colloid follicles and mostly observed towards the ventral margin of the PD. Sometimes disruption of the outer margin with depletion of fibrous material from the cavity was observed. Hormone-secreting cells of various types, anti-adrenocorticotropic-hormone-, anti-prolactin-, anti-growth-hormone- and anti-luteinizing-hormone-immunoreactive cells were encountered bordering both the colloid follicles and fibrous cysts. Neither the colloid nor the fibrous material showed any immunoreaction to any of the pituitary hormone antisera. On histochemical staining colloid was positive to periodic acid-Schiff (PAS) and fibrous materials stained with Alcian blue-PAS-orange G staining. Colloid depositions in the pituitary gland of these two wild birds were correlated to age, more in numbers in the adult birds than in the young ones. Fibrous-material-containing cysts were elucidated in the pituitary gland of adult birds only. These were more prevalent in the pituitary of reproductively active birds. Regular morphology of the colloid follicles, overall distribution in the adenohypophysis and dense nature of deposition of the colloid suggest the accumulation of this type may be the secretory products of both granulated and agranulated pituitary cell types. Absence of immunoreactivity of the colloid against pituitary hormone antisera points out that the storage form may differ chemically from the bioactive hormones. The spatial distribution of fibrous-material-containing cysts mostly towards the ventral PD, observations of immunoreactive cell fragmentations inside the cysts, and their disrupted margins suggest these structures may have some role in discharging the intraglandular degradation products. 2006 S. Karger AG, Basel

Inadvertent trypan blue posterior capsule staining during cataract surgery.

PubMed

Burkholder, Bryn M; Srikumaran, Divya; Nanji, Afshan; Lee, Bryan; Weinberg, Robert S

2013-04-01

To report 5 cases of inadvertent posterior capsule staining with trypan blue during phacoemulsification. Retrospective, observational case series. Five cases of posterior capsule staining with trypan blue were identified from cataract surgeries performed at an academic institution. All 5 eyes underwent phacoemulsification with use of iris retractors. The surgical videos from each case were reviewed to better understand the mechanisms and risk factors for posterior capsule staining with trypan blue and techniques to avoid this complication. No eyes had clinical evidence of zonular pathology during their preoperative examination. Only 1 patient reported a possible childhood history of trauma to both eyes. One eye had uveitis, requiring posterior synechialysis. All 5 cases involved the use of iris retractors. No other intraoperative complications occurred, and the intraocular lens was successfully placed in the capsular bag in all cases. All eyes had resolution of posterior capsule staining by postoperative day 8. Inadvertent posterior capsule staining with trypan blue can occur in eyes that appear structurally normal. The use of iris retractors may facilitate posterior capsule staining by allowing the posterior flow of trypan blue under the iris and through the zonules to the posterior capsule. Surgeons should consider techniques to minimize the risk of posterior capsule staining, particularly in cases involving the use of iris retractors. Copyright © 2013 Elsevier Inc. All rights reserved.

Better detection of Demodex mites by Löffler's alkaline methylene blue staining in patients with blepharitis.

PubMed

Kiuchi, Katsuji

2018-01-01

To determine whether the Löffler's alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution ( P <0.01, Wilcoxon test). The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation.

Better detection of Demodex mites by Löffler’s alkaline methylene blue staining in patients with blepharitis

PubMed Central

Kiuchi, Katsuji

2018-01-01

Purpose To determine whether the Löffler’s alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Materials and methods Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. Results The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution (P<0.01, Wilcoxon test). Conclusion The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation. PMID:29713140

Secondary sea-blue histiocytosis derived from Niemann-Pick disease.

PubMed

Suzuki, Osamu; Abe, Masafumi

2007-04-01

Sea-blue histiocytosis is a rare disorder seen in patients with lipid metabolic or ceroid storage diseases. Sea-blue histiocytes are ceroid-laden macrophages detectable by May-Giemsa staining. We report a case of a 28-year-old woman diagnosed with Niemann-Pick disease at 2 or 3 years of age. To confirm this diagnosis, we examined her bone marrow, which revealed scattered foci containing aggregates of foamy macrophages. May-Giemsa staining identified blue-staining foamy macrophages, referred to as sea-blue histiocytes. In summary, we report the detection of sea-blue histiocytosis in an adult with Niemann-Pick disease.

Baicalein inhibits pulmonary carcinogenesis-associated inflammation and interferes with COX-2, MMP-2 and MMP-9 expressions in-vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandrashekar, Naveenkumar; Selvamani, Asokkumar; Subramanian, Raghunandhakumar

2012-05-15

The objective of the present study is to investigate the therapeutic efficacy of baicalein (BE) on inflammatory cytokines, which is in line with tumor invasion factors and antioxidant defensive system during benzo(a)pyrene [B(a)P] (50 mg/kg body weight) induced pulmonary carcinogenesis in Swiss albino mice. After experimental period, increased levels of total and differential cell count in bronchoalveolar lavage fluid were observed. Accompanied by marked increase in immature mast cell by toluidine blue staining and mature mast cell by safranin–alcian blue staining in B(a)P-induced lung cancer bearing animals. Protein expression levels studied by immunohistochemistry and immunoblot analysis of cytokines such asmore » tumor necrosis factor-α, interleukin-1β and inducible nitric oxide synthase were also found to be significantly increased in lung cancer bearing animals. B(a)P-exposed mice lung exhibits activated expression of nuclear transcription factor kappa-B as confirmed by immunofluorescence and immunoblot analysis. Administration of BE (12 mg/kg body weight) significantly counteracted all the above deleterious changes. Moreover, assessment of tumor invasion factors on protein levels by immunoblot and mRNA expression levels by RT-PCR revealed that BE treatment effectively negates B(a)P-induced upregulated expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and cyclo-oxygenase-2. Further analysis of lipid peroxidation markers such as thiobarbituric acid reactive substances, hydro-peroxides and antioxidants such as glutathione-S-transferase and reduced glutathione in lung tissue was carried out to substantiate the antioxidant effect of BE. The chemotherapeutic effect observed in the present study is attributed to the potent anti-inflammatory and antioxidant potential by BE against pulmonary carcinogenesis. -- Highlights: ► BE treatment protects from inflammatory cells and mast-cells accumulation in lungs. ► BE altered the expressions of TNF-α, IL-1β, i-NOS and NF-κBp65 at protein levels. ► BE modulates the expressions of MMP-2, MMP-9 and COX-2 at protein and mRNA levels. ► BE decreases LPO levels and enhances antioxidant status.« less

The Immunohistochemistry and Toluidine Blue Roles for Helicobacter pylori Detection in Patients with Gastritis

PubMed Central

Tajalli, Raziye; Nobakht, Maliheh; Mohammadi-Barzelighi, Hajar; Agah, Shahram; Rastegar-Lari, Abdolaziz; Sadeghipour, Alireza

2013-01-01

Background: Helicobacter pylori, which is associated with many upper gastrointestinal diseases, is found in half of the population of the world. Several special stains and immunohistochemistry stain for H. pylori are available. The need for and usefulness of immunohistochemical (IHC) technique has been debated for years. Toluidine blue is a simple stain for microbiological studies and is easily available in laboratories. Therefore, this study was conducted to compare hematoxylin and eosin (H&E), Giemsa and toluidine blue staining with immunehistochemistry for detection of H. pylori in patients with gastritis and also to correlate the results of these staining methods with pathological grading. Methods: We reviewed 54 consecutive gastric biopsy specimens stained by H&E and Giemsa as well as by toluidine blue and immunohistochemistry stains for H. pylori. Results: H. pylori was positively identified by IHC in 43 (79.63%) patients, while positive samples were found in 18 (33.33%), 24 (44.44%) and 33 (61.11%) patients using H&E, Giemsa and toluidine blue staining methods. Our results showed that classical histological staining methods are not sensitive enough to identify low numbers or coccoid forms of organism, while toluidine blue and immunohistochemistry play an important role in detection of H. pylori infection. Conclusion: Toluidine blue has been proved to be much more reliable than H&E and Giemsa in detection of H. pylori. In addition, in post treatment biopsies and in biopsies with unexplained chronic active gastritis without histological evidence of H. pylori should have immunohistochemistry done to detect possible low density or coccoid form of organisms. PMID:23279833

Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue.

PubMed

Inaga, Sumire; Hirashima, Sayuri; Tanaka, Keiichi; Katsumoto, Tetsuo; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2009-07-01

The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x40 to x10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.

Enteric neuromodulators and mucus discharge in a fish infected with the intestinal helminth Pomphorhynchus laevis.

PubMed

Bosi, Giampaolo; Shinn, Andrew Paul; Giari, Luisa; Sayyaf Dezfuli, Bahram

2015-07-08

In vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge. A modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue - periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells. From a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host(-1), mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship. The presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.

PubMed

McCrone, E L; Lucey, D R; Weller, P F

1988-11-10

To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially present with eosinophils did not fluoresce with aniline blue. The fluorescent staining eosinophils within filters provided bright, non-fading images that facilitated visual microscopic counting and were of sufficiently high contrast, unlike those with conventional eosinophil stains, to allow image analyzer based enumeration of eosinophil chemotactic responses at levels through the filters. Although not cell type-specific, congo red and ethidium bromide also provided high contrast, fluorescent images of all leukocyte types within chemotactic filters. Fluorescent staining with aniline blue constitutes a rapid, stable and eosinophil-specific stain that facilitates the visual or image analyzer-based quantitation of eosinophil chemotaxis.

Differentiating vulvar intraepithelial neoplasia from nonneoplastic epithelial disorders. The toluidine blue test.

PubMed

Joura, E A; Zeisler, H; Lösch, A; Sator, M O; Müllauer-Ertl, S

1998-08-01

To determine the effectiveness of the toluidine blue test in the differentiation of vulvar intraepithelial neoplasia (VIN) and nonneoplastic epithelial disorders (NNEDs). This retrospective clinical study included all women with VIN (n = 24) and NNED (n = 72) referred to a vulvar clinic at a university hospital during a two-year period. Vulvoscopy, staining of vulvar epithelium with 1% toluidine blue and punch biopsy were performed. Vulvar epithelium demonstrated toluidine blue staining in 100% of the patients with VIN 3, in 83% of women with VIN 1-2, in 50% of the women with squamous cell hyperplasia and in 10% of the women with lichen sclerosus. The differences in staining between the groups were statistically significant (P < .001). The sensitivity of toluidine blue staining for the detection of VIN was 92%; the negative predictive value 96% in teh investigated cohort. The specificity for strong staining was 88%. The toluidine blue test is an inexpensive and reliable method of separating VIN from hyperplastic NNED areas and choosing a biopsy site on the vulva.

Reproduction of the salamander Siren intermedia le conte with especial reference to oviducal anatomy and mode of fertilization.

PubMed

Sever, David M; Rania, Lisa C; Krenz, John D

1996-03-01

Reproduction was studied in a South Carolina population of the paedomorphic salamander Siren intermedia with emphasis on anatomy of the female oviduct. The oviduct forms 67-79% of the snout-vent length in this elongate species and can be divided into three portions. The atrium, 7-13% of oviducal length, is the narrow anteriormost portion, with the ostial opening immediately caudad of the transverse septum. The ampulla, 63-75% of oviducal length, is the highly convoluted, middle portion in which gelatinous coverings are added to the eggs during their passage. Hypertrophy of the oviducal glands in the ampulla causes the ampulla to increase in diameter during the ovipository season. The secretion of the eosinophilic oviducal glands is intensely positive following staining with the periodic acid-Schiff procedure and does not react with alcian blue at pH 2.5. This staining reaction, coupled with the presence of abundant rough endoplasmic reticulum and Golgi complexes, indicates that the secretion contains a glycoprotein. The ovisac, 16-25% of oviducal length, is the most posterior portion of the oviduct and holds up to 10-11 eggs prior to oviposition. Oviducal glands similar to those in the ampulla are absent in the ovisac. Oviposition in female sirens occurs during February-April in this population, and male spermiation is concurrent. Entire oviducts were sectioned from three females collected during the ovipository season and from two collected prior to the breeding season, and sperm were not found in the oviducts of these specimens. Thus no evidence was found for internal fertilization or sperm storage in the oviducts of sirens. © 1996 Wiley-Liss, Inc. Copyright © 1996 Wiley-Liss, Inc.

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing.

PubMed

Kshersagar, Jeevitaa; Kshirsagar, Ravi; Desai, Shashikant; Bohara, Raghvendra; Joshi, Meghnad

2018-03-05

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin-eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion's trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco's modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients' needs.

FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice.

PubMed

Marongiu, Mara; Marcia, Loredana; Pelosi, Emanuele; Lovicu, Mario; Deiana, Manila; Zhang, Yonqing; Puddu, Alessandro; Loi, Angela; Uda, Manuela; Forabosco, Antonino; Schlessinger, David; Crisponi, Laura

2015-07-02

Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation. Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR. Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus. Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.

Effect of low level laser and low intensity pulsed ultrasound therapy on bone remodeling during orthodontic tooth movement in rats.

PubMed

Alazzawi, Mohammed Mahmood Jawad; Husein, Adam; Alam, Mohammad Khursheed; Hassan, Rozita; Shaari, Rumaizi; Azlina, Ahmad; Salzihan, M S

2018-04-16

Quality bone regeneration, which leads to the improvement of bone remodeling, is essential for orthodontic treatment. In order to improve bone regeneration and increase the amount of tooth movement, different techniques have been implemented. The object of this study is to compare the effects of low-level laser therapy (LLLT), low-intensity pulsed ultrasound (LIPUS), and their combination on bone remodeling during orthodontic tooth movement. Eighty (80) male, 6-week-old Sprague Dawley rats were grouped in to four groups, the first group was irradiated with (940 nm) diode laser, second group with LIPUS, and third group with combination of both LLLT and LIPUS. A forth group used was a control group in an incomplete block split-mouth design. The LLLT and LIPUS were used to treat the area around the moving tooth once a day on days 0-7, then the experiment was ended in each experimental endpoint (1, 3, 7, 14, and 21 days). For amount of tooth movement, models were imaged and analyzed. Histological examination was performed after staining with (hematoxylin and eosin) and (alizarin red and Alcian Blue) stain. One step reverse transcription-polymerase chain reaction RT-PCR was also performed to elucidate the gene expression of RANK, RANKL, OPG, and RUNX-2. The amount of tooth movement, the histological bone remodeling, and the RT-PCR were significantly greater in the treatment groups than that in the control group. Among the treatment groups, the combination group was the highest and the LIPUS group was the lowest. These findings suggest that LLLT and LIPUS can enhance the velocity of tooth movement and improve the quality of bone remodeling during orthodontic tooth movement.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

PubMed

Pelle, Dominic W; Peacock, Jacqueline D; Schmidt, Courtney L; Kampfschulte, Kevin; Scholten, Donald J; Russo, Scott S; Easton, Kenneth J; Steensma, Matthew R

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Genetic and Functional Studies of the Intervertebral Disc: A Novel Murine Intervertebral Disc Model

PubMed Central

Pelle, Dominic W.; Peacock, Jacqueline D.; Schmidt, Courtney L.; Kampfschulte, Kevin; Scholten, Donald J.; Russo, Scott S.; Easton, Kenneth J.; Steensma, Matthew R.

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration. PMID:25474689

Non-invasive in vivo molecular imaging of intra-articularly transplanted immortalized bone marrow stem cells for osteoarthritis treatment.

PubMed

Peng, Bou-Yue; Chiou, Chi-Sheng; Dubey, Navneet Kumar; Yu, Sung-Hsun; Deng, Yue-Hua; Tsai, Feng-Chou; Chiang, Han-Sun; Shieh, Ying-Hua; Chen, Wei-Hong; Deng, Win-Ping

2017-11-14

Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.

[Subchondral drilling method combined with gum-bletilla complex to repair articular cartilage defects].

PubMed

Huang, Yong; Wang, Xin-Ling; Qiu, Heng; Xiao, Yi-Cheng; Wu, Zong-Hong; Xu, Jian

2018-02-01

Two types(A model and B model) of articular cartilage defect models were prepared by using adult New Zealand white rabbits. A model group was applied by drilling without through subchondral bone, whose right joint was repaired by composite scaffolds made by seed cell, gum-bletilla as well as Pluronic F-127, and left side was blank control. B model group was applied by subchondral drilling method, whose right joint was repaired by using composite scaffolds made by gum-bletilla and Pluronic F-127 without seed cells, and left side was blank control. Autogenous contrast was used in both model types. In addition, another group was applied with B model type rabbits, which was repaired with artificial complex material of Pluronic F-127 in both joint sides. 4, 12 and 24 weeks after operation, the animals were sacrificed and the samples were collected from repaired area for staining with HE, typeⅡcollagen immunohistochemical method, Alcian blue, and toluidine blue, and then were observed with optical microscope. Semi-quantitative scores were graded by referring to Wakitanis histological scoring standard to investigate the histomorphology of repaired tissue. Hyaline cartilage repairing was achieved in both Group A and Group B, with satisfactory results. There were no significant differences on repairing effects for articular cartilage defects between composite scaffolds made by seed cell, gum-bletilla and Pluronic F-127, and the composite scaffolds made by gum-bletilla and Pluronic F-127 without seed cell. Better repairing effects for articular cartilage defects were observed in groups with use of gum-bletilla, indicating that gum-bletilla is a vital part in composite scaffolds material. Copyright© by the Chinese Pharmaceutical Association.

Prenatal development of the normal human vertebral corpora in different segments of the spine.

PubMed

Nolting, D; Hansen, B F; Keeling, J; Kjaer, I

1998-11-01

Vertebral columns from 13 normal human fetuses (10-24 weeks of gestation) that had aborted spontaneously were investigated as part of the legal autopsy procedure. The investigation included spinal cord analysis. To analyze the formation of the normal human vertebral corpora along the spine, including the early location and disappearance of the notochord. Reference material on the development of the normal human vertebral corpora is needed for interpretation of published observations on prenatal malformations in the spine, which include observations of various types of malformation (anencephaly, spina bifida) and various genotypes (trisomy 18, 21 and 13, as well as triploidy). The vertebral columns were studied by using radiography (Faxitron X-ray apparatus, Faxitron Model 43,855, Hewlett Packard) in lateral, frontal, and axial views and histology (decalcification, followed by toluidine blue and alcian blue staining) in and axial view. Immunohistochemical marking with Keratin Wide Spectrum also was done. Notochordal tissue (positive on marking with Keratin Wide Spectrum [DAKO, Denmark]) was located anterior to the cartilaginous body center in the youngest fetuses. The process of disintegration of the notochord and the morphology of the osseous vertebral corpora in the lumbosacral, thoracic, and cervical segments are described. Marked differences appeared in axial views, which were verified on horizontal histologic sections. Also, the increase in size was different in the different segments, being most pronounced in the thoracic and upper lumbar bodies. The lower thoracic bodies were the first to ossify. The morphologic changes observed by radiography were verified histologically. In this study, normal prenatal standards were established for the early development of the vertebral column. These standards can be used in the future--for evaluation of pathologic deviations in the human vertebral column in the second trimester.

Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro.

PubMed

Ying, Xiaozhou; Zhang, Wei; Cheng, Shaowen; Nie, Pengfei; Cheng, Xiaojie; Shen, Yue; Wang, Wei; Xue, Enxing; Chen, Qingyu; Kou, Dongquan; Peng, Lei; Zhang, Yu; Lu, Chuanzhu

2012-11-01

Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5) M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5) M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6) M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P < 0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

Preference of Formosan subterranean termites for blue-stained southern yellow pine sapwood.

PubMed

Little, N S; Blount, N A; Londo, A J; Kitchens, S C; Schultz, T P; McConnell, T E; Riggins, J J

2012-10-01

Little research has been conducted to investigate interactions between the invasive Formosan subterranean termite, Coptotermes formosanus Shiraki, and pine bark beetles native to the southeastern United States. Facilitative interactions between these organisms could alter stand dynamics and impact wood utilization strategies. American Wood Protection Association Standard E1-09 choice tests were carried out to determine the feeding preference of Formosan subterranean termites for blue-stained versus unstained southern yellow pine sapwood. Three separate colonies of Formosan subterranean termites consumed on average twice as much air-dried blue-stained southern yellow pine sapwood over unstained air-dried controls. Additionally, Formosan subterranean termites consumed over five-times more kiln-dried blue-stained sapwood than unstained kiln-dried control wafers. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, and the utilization of blue-stained southern pine building products in the southeastern United States, where Formosan subterranean termites have become established.

Methylene blue-aided cholangioscopy in patients with biliary strictures: feasibility and outcome analysis.

PubMed

Hoffman, A; Kiesslich, R; Bittinger, F; Galle, P R; Neurath, M F

2008-07-01

Chromoendoscopy using methylene blue is employed in the gastrointestinal tract to delineate neoplastic lesions. We tested the value of chromoendoscopy during choledochoscopy for characterization of local inflammation, neoplasias, and other alterations in patients with biliary strictures. Patients with suspected biliary lesions were scheduled for endoscopic retrograde cholangiography with subsequent cholangioscopy. After initial inspection of the bile duct, 15 ml methylene blue (0.1 %) was administered via the working channel of the cholangioscope. Newly appearing circumscribed or unstained lesions were judged according to their macroscopic type and staining features. Methylene-blue-aided diagnosis was compared with either clinical follow-up of the patients or, in some cases, with the results of targeted biopsies. A total of 55 patients [biliary stenosis/cholestasis of unknown origin (n = 24), stenosis after orthotopic liver transplantation (n = 11), primary sclerosing cholangitis (n = 20)] were included. Methylene blue unmasked subtle mucosal changes and permitted macroscopic characterization of circumscribed lesions. Characteristic surface staining patterns were seen in chronic inflammation, dysplasia, and ischemic-type biliary lesions. Nondysplastic mucosa appeared homogeneously stained, whereas scarred strictures showed a weak uptake of methylene blue. In this prospective feasibility study, methylene-blue-aided cholangioscopy was used for the first time to define different staining patterns of the bile duct. The differences in staining patterns identified normal, dysplastic, and inflamed mucosa of the bile duct, as was proved by follow-up or, in some cases, histology. Whereas homogeneous staining predicted the presence of normal mucosa, absence of staining of circumscribed lesions, or diffused staining of such lesions, represented neoplastic changes or inflammation.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Goblet Cells and Mucus Types in the Digestive Intestine and Respiratory Intestine in Bronze Corydoras (Callichthyidae: Teleostei).

PubMed

Leknes, I L

2015-10-01

The structure and histochemical properties of the intestine in bronze corydoras (Corydoras aeneus), a stomach-containing teleost, are described, with emphasis on goblet cells and mucin types. The proximal intestine displayed a normal structure for teleosts, whereas the distal intestine was wide, translucent, thin-walled, richly vascularized and constantly filled with air, suggesting an important respiratory role. Goblet cells were common throughout the entire intestine and displayed a variable, but mainly faint metachromatic colour after toluidine blue. They were moderately coloured by alcian blue at both pH 2.5 and 0.2 and displayed no colour after periodic acid followed by Schiff's solution (PAS), but a distinct purple-brown colour after high iron diamine followed by alcian blue (pH 2.5). Together, these results suggest that the mucin in the intestine goblet cells consists mainly of sulphated proteoglycans. Further, the results from the present lectin and neuraminidase tests suggest that these mucins contain much N-acetylglucoseamines and some N-acetylgalactosamines and sialic acid, but seem to lack glucose and mannose. They also contain some galactose-N-acetylgalactosamines sequences, normally hidden by sialic acid. The distinct brush border and mucus layer on the epithelial cells in the respiratory intestine may indicate some digestive roles, such as absorption of water, ions and simple carbohydrates. As sulphated proteoglycans are tough and attract much water, this mucus may play important roles in the protection against mechanical and chemical damages and in the defence against micro-organisms throughout the entire intestine, but in the respiratory intestine it may impede significantly the oxygen uptake. However, as this part of the intestine usually contains no digesta, but is completely filled with air, frequently renewed by dry air from the atmosphere, and the main function of the mucus may be to protect the respiratory epithelium against a destroying and dangerous desiccation. © 2014 Blackwell Verlag GmbH.

[Histopathological and immunohistochemical studies on mucous cysts].

PubMed

Kuroda, N

1989-01-01

The present study investigated the histopathology, histochemistry of mucopolysaccharides, and immunohistochemistry of oral mucous cysts. The materials were obtained from ninety cases that were histopathologically diagnosed as oral mucous cysts at the Department of Oral Pathology, Meikai University School of Dentistry. Mucopolysaccharide staining was done with PAS, alcian blue (AB, pH 2.5) and high iron diamine (HID). Immunohistochemical studies were focused on secretory component (SC), lactoferrin (Lf), alpha-amylase (Am), IgA, lysozyme (Ly), and keratin (Kr). The following results were obtained: 1. Histopathological findings. (1) Retention and/or retention-like type cysts occurred in was twenty-six cases and the extravasation type in sixty-four cases. (2) Cases showing epithelial lining of the cystic wall were only eight in number, and many cystic walls were contained granulation tissue (fifty cases). (3) As for inflammation of the cystic wall, the degree was slight, and infiltrated cells were mainly macrophages (so-called mucinophages) and lymphocytes. (4) Regarding adjoining salivary glands, acinar cells showed atrophic changes, and hypertrophy of mucous acinar cells was evident. Many ducts showed dilatation, and stromal connective tissue showed fibrosis and hyalinization. 2. Histochemical findings on mucopolysaccharides. (1) Mucous materials in cystic cavity, mucous acinar cells, and secretory materials in ductal lumens were intensely stained by PAS and AB. But stainability with AB was less than that with PAS staining. Serous acinar cells and ductal epithelium were negative to PAS and AB staining. (2) Stainability of the above with HID was less than at with PAS or AB. Cystic walls were not stained by HID. Mucous acinar cells reactive with HID were intensely stained, but the number of the positive cells was limited when compared with the numbers of PAS-and AB-positive cells. 3. Immunohistochemical findings. (1) As for mucous materials in the cystic cavity, reactions for Sc, Am, IgA, and Ly were all positive, whereas those for Lf and Kr were negative. (2) Staining of cystic walls was generally weak: the walls were negative for IgA, Ly and Kr showed and borderline or slightly positive for Sc, Lf, and Am. (3) Mucous acinar cells were negative for all markers examined in this study, but serous acinar cells and/or demilunes were markedly positive for Sc, Ly, and Am. (4) In ductal epithelial cells, Ly and Kr were negative, but IgA was borderline or slightly positive. Sc and Am gave intensely positive staining. (5) Secretory materials in ductal lumens, Sc were intensely positive for Lf, and Ly; slightly or moderately positive for IgA; and slightly positive or negative for Am.(ABSTRACT TRUNCATED AT 400 WORDS)

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis.

PubMed

Bonnell, B S; Larabell, C; Chandler, D E

1993-06-01

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a three-dimensional network of interconnected fibers extending nearly 50 microns from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a three-dimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50-60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix.

Celiac disease and intestinal metaplasia of the esophagus (Barrett's esophagus).

PubMed

Maieron, Roberto; Elli, Luca; Marino, Marco; Floriani, Irene; Minerva, Francesco; Avellini, Claudio; Falconieri, Giovanni; Pizzolitto, Stefano; Zilli, Maurizio

2005-01-01

Previous studies on celiac patients demonstrated that exposure to gliadin alters the motility of the upper gastrointestinal tract, leading to increased acid reflux. No literature is available regarding the possible presence of specialized intestinal metaplasia of the esophagus as a consequence of chronic reflux in adult celiac patients. Our purpose was to evaluate endoscopically and histologically the esophagi of a group of untreated celiac patients. We studied 60 celiac patients, 13 men and 47 women (mean age, 40 +/- 14 [SD] years; range, 18-80 years), at their first endoscopy (following a normal diet). The distal esophagus was evaluated and multiple biopsies were taken. Hematoxylin-eosin and alcian blue stainings were performed. A group of nonceliac, age- and sex-matched patients was used as a control. We found intestinal metaplasia in the distal esophagus of 16 of 60 (26.6%) celiacs (mean age, 45 +/- 13 years; range, 27-75 years), in comparison with a control-group prevalence of 10.9% (OR, 3.9; 95% CI, 1.4-11.2%). Among the celiac group with metaplasia, only one patient had reflux-like symptoms. None had esophagitis. In conclusion, we observed an increased prevalence of esophageal metaplasia in patients with celiac disease. This finding could be the result of motor abnormalities leading to chronic acid reflux, combined with a mucosa which is sensitive to gliadin.

Glycopattern analysis of acidic secretion in the intestine of the red-eared slender turtle; Trachemys scripta elegans (Testudines: Emydidae).

PubMed

Scillitani, Giovanni; Mentino, Donatella; Mastrodonato, Maria

2017-10-01

The secretion of the goblet cells in the intestine of Trachemys scripta elegans was studied in situ by histochemical methods to analyze the diversity of sugar chains, with particular regard to the acidic glycans. Conventional histochemical stains (Periodic acid-Schiff, Alcian Blue pH 2.5, High Iron Diamine) and binding with ten FITC-labelled lectins combined with chemical and enzymatic pre-treatments were used to characterize the oligosaccharidic chains. The intestine can be divided into three regions, i.e. a duodenum, a small intestine and a large intestine. Goblet cells were observed in all the three tracts and presented an acidic secretion. WGA, LFA, PNA and SBA binding was observed only after desulfation. Glycans secreted by the three tracts consist mainly of sulfosialomucins with 1,2-linked fucose, mannosylated, glucosaminylated and subterminal galactosyl/galactosaminylated residuals. Differences among tracts are quantitative rather than qualitative, with sulfated, galactosaminylated and glycosaminylated residuals increasing from duodenum to large intestine, and galactosylated and fucosylated residuals showing an opposite trend. Variation is observed also between apices and bases of villi in both duodenum and small intestine, where sulphation decreases from the base to the apex and glycosylation shows an opposite trend. Functional implication of these findings is discussed in a comparative context. Copyright © 2017 Elsevier Ltd. All rights reserved.

Specificity of dermal mucin in the diagnosis of lupus erythematosus: comparison with other dermatitides and normal skin.

PubMed

Vincent, Jeremy G; Chan, May P

2015-10-01

Increased dermal mucin is a feature of lupus erythematosus (LE); however, its amount and distribution have not been well characterized. The differentiation of LE from other forms of dermatitis can be challenging when other features of LE are subtle or equivocal. One hundred and thirty-five skin specimens showing LE, graft vs. host disease, erythema multiforme/fixed drug eruption, lichen planus, polymorphous light eruption (PMLE), urticaria, eczematous dermatitis and psoriasis and normal skin with and without photodamage were collected. The amounts of mucin in the papillary, superficial reticular and deep reticular dermis were scored from 0 to 3 on hematoxylin-eosin (H&E) and alcian blue (AB) stains, and compared between groups. The mean scores in the reticular dermis were significantly higher in LE than in other categories except PMLE and eczematous dermatitis. A combined H&E + AB score of ≥5 in the superficial reticular dermis gave an overall specificity of 85.7% for LE. Mucin in the papillary dermis failed to distinguish among entities. Normal photodamaged skin showed significantly more mucin in the superficial reticular dermis compared to non-photodamaged skin. While LE is associated with increased mucin deposition, scant to moderate amount of mucin alone has limited specificity and is common in other dermatitides or photodamaged skin. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Penicillin reduces eustachian tube gland tissue changes in acute otitis media.

PubMed

Andersen, Henrik; Thomsen, Jens; Cayé-Thomasen, Per

2005-08-01

The volume of the mucous paratubal glands and the number of the mucus-producing goblet cells in the middle ear and Eustachian tube (ET) are increased after experimental acute otitis media (AOM). The present investigation examines a potential effect of penicillin on the changes in goblet cell density and gland structures of the ET during and after AOM. Middle ear inoculation of Streptococcus pneumoniae in 50 rats. Two days later, 25 rats were given penicillin V as one daily dose for 5 days. Twenty-five rats received no treatment. Five animals from each group were sacrificed on days 4, 8, 16, 90, and 180. The ET was dissected and decalcified, followed by paraffin embedding, serial transverse sectioning, and PAS/alcian blue staining. The goblet cell density and the paratubal gland composition and volume were determined in every 20th section, using a light microscope. Penicillin reduced the increase of goblet cell density from day 8 and through 6 months, whereas the increase of the paratubal mucous gland volume was unaffected by treatment. We conclude that penicillin reduces the increase of ET goblet cell density during and after acute otitis media, whereas the paratubal gland volume remains unaffected. An increased mucosal secretory capacity and indicated excessive secretion of mucus may contribute to the deteriorated ET function found after AOM and thus predispose, sustain, or aggravate middle ear disease. This may be prevented by penicillin treatment.

Cytologically atypical anal sac adenocarcinoma in a dog.

PubMed

Sakai, Hiroki; Murakami, Mami; Mishima, Hiroyuki; Hoshino, Yuki; Mori, Takashi; Maruo, Kohji; Yanai, Tokuma

2012-06-01

A 10-year-old intact female Shetland Sheepdog with tenesmus had a subcutaneous mass at the left ventral aspect of the anus. On cytologic examination, 2 types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Eosinophilic PAS- and Alcian blue-positive secretory material was found in the center of some glandular structures. Both neoplastic cell types had positive staining with paradoxical concanavalin A and expressed cytokeratin, but not vimentin, S-100, α-smooth muscle actin, or desmin. Based on location and histologic and immunohistochemical features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal sac of dogs. © 2012 American Society for Veterinary Clinical Pathology.

Study on Mucin in Normal-Appearing Leg Skin.

PubMed

Fernandez-Flores, Angel

2017-03-01

Dermal deposits of mucin in the legs have been described associated with venous insufficiency. However, some degree of stasis dermatitis is generally common in aged individuals. Therefore, some amount of mucin is expected a priori in the reticular dermis of aged patients, even in the absence of clinical lesions. To test this hypothesis, the authors investigated the mucin in the legs of aged individuals without any dermatologic disease. Cutaneous samples were taken from the legs of 15 autopsy cases. A sample of the skin of the legs (either from the left or the right leg without any distinction being made) was randomly taken (without selecting any specific area or attending to macroscopical features). The skin samples were fixed in formaldehyde, and sections obtained from all samples were stained with hematoxylin and eosin, iron, and Alcian blue. Iron deposits were graded as 0/4 in 7 cases, as 1/4 in 4 cases, as 2/4 in 2 cases, and as 4/4 in 2 cases. Cases with greater deposits of iron also had other signs of stasis, such as neovascularization. All the samples scored 0 for dermal mucin deposits in the reticular dermis. The authors conclude that mucin deposits in the legs are not inherent to aging. Therefore, any mucin deposit in the reticular dermis, as well as expansion of the periadnexal dermis by mucin deposits, should be considered abnormal.

Development of short-snouted seahorse (Hippocampus hippocampus, L. 1758): osteological and morphological aspects.

PubMed

Novelli, B; Otero-Ferrer, F; Socorro, J A; Caballero, M J; Segade-Botella, A; Molina Domínguez, L

2017-06-01

Information about early development after male release lags behind studies of juveniles and adult seahorses, and newborn seahorses, similar in shape to adults, are considered juveniles or fry. During early life, Hippocampus hippocampus present behavioural (shift in habitat, from planktonic to benthic) and morphological changes; for this reasons, the aims of this study are to define the stage of development of H. hippocampus after they are expelled from the male brood pouch and to establish direct or indirect development through an osteological analysis. The ossification process was studied in 120 individuals, from their release to 30 days after birth. To analyse the osteological development, Alcian Blue-Alizarin Red double staining technique for bone and cartilage was adapted to this species. At birth, H. hippocampus presents a mainly cartilaginous structure that ossifies in approximately 1 month. The bony armour composed of bony rings and plates develops in 10 days. The caudal fin, a structure absent in juveniles and adult seahorses, is present at birth and progressively disappears with age. The absence of adult osteological structure in newborns, like coronet, bony rings and plates, head spines and components allowing tail prehensile abilities, suggests a metamorphosis before the juvenile stage. During the indirect development, the metamorphic stage started inside brood pouch and followed outside and leads up to reconsider the status of H. hippocampus newborns.

Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells.

PubMed

Ahmadyan, Sorour; Kabiri, Mahboubeh; Hanaee-Ahvaz, Hana; Farazmand, Ali

2018-06-01

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550 mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.

Two T7-like Bacteriophages, K5-2 and K5-4, Each Encodes Two Capsule Depolymerases: Isolation and Functional Characterization.

PubMed

Hsieh, Pei-Fang; Lin, Hsiao-Hsuan; Lin, Tzu-Lung; Chen, Yi-Yin; Wang, Jin-Town

2017-07-04

Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.

Sustained methylene blue staining to guide anatomic hepatectomy for hepatocellular carcinoma: Initial experience and technical details.

PubMed

Shou-wang, Cai; Shi-zhong, Yang; Wen-ping, Lv; Geng, Chen; Wan-qing, Gu; Wei-dong, Duan; Wei-yi, Wang; Zhi-qiang, Huang; Jia-hong, Dong

2015-07-01

The boundary of the target hepatic segment within the liver parenchyma cannot be marked by the use of a conventional anatomic hepatectomy approach. This study describes a novel methylene blue staining technique for guiding the anatomic resection of hepatocellular carcinoma (HCC). Between February 2009 and February 2012, anatomic hepatectomy was performed in 106 patients with HCC via a novel, sustained methylene blue staining technique. Sustained staining was achieved by injecting methylene blue into the distal aspect of the portal vein after exposing Glisson's sheath. The hepatic pedicle was immediately ligated, and the hepatic parenchymal transection was performed along the interface between methylene blue stained tissue and unstained tissue. Anatomic hepatectomies included subsegmentectomy (n = 16), monosegmentectomy (n = 57), multisegmentectomy (n = 27), and hemihepatectomy (n = 6). The portal vein was injected successfully with methylene blue in 100% of cases, and complete staining of the target hepatic segment was achieved in 98 of 106 (92.5%) cases. Mean intraoperative bleeding was 360 ± 90 mL, and the postoperative complication rate was 24.5% (26/106). No perioperative mortality occurred. Operative margins were all negative on pathologic examination. Mean duration of postoperative follow-up was 40 months (range, 24-60). No local recurrence (around the operative margin) occurred. This novel technique of achieving sustained staining by injecting methylene blue then immediately ligating the hepatic pedicle is simple and feasible. It can guide the selection of the operative margin during hepatic parenchyma transection to improve the accuracy of anatomic hepatectomy for the treatment of HCC. Copyright © 2015 Elsevier Inc. All rights reserved.

Toluidine blue: rapid and simple malaria parasite screening and species identification.

PubMed

Awale, Rupali; Maji, Ratnaprabha; Patil, Parag; Lingiah, Raghavendra; Mukhopadhyay, Ashok Kumar; Sharma, Subhadra

2017-01-01

Malaria, a febrile illness mostly confined to the tropical countries is transmitted by bite of infected female Anopheles mosquito. In 2015 alone, 88% of the malaria burden and 90% deaths due to malaria were confined to the African and Asian countries. Although number of tests are available for rapid diagnosis and screening for malaria, peripheral blood smear examination remains the gold standard. Leishman stain is recommended by WHO however herein we evaluate one of the alternative methods of staining which is simple and rapid. Fifty patients attending the various outpatient departments of the tertiary care hospital with fever and suspected to have malaria were selected. Two thin-air dried smears prepared from the peripheral venous blood from these subjects were stained by Leishman and Toluidine blue method. The findings of the slides by two independent qualified professionals were noted and the results were analyzed. A total of 14% (7/50) cases were diagnosed to have malaria. All the malaria cases which were positive in Leishman stain were also detected in Toluidine blue stain. Malarial parasites were clearly visible against the homogenously light green background in Toluidine blue. The detection of malarial parasite by Toluidine blue was quick, easy and confirmative. Toluidine blue stained peripheral blood smear allows for easy identification and speciation of malarial parasite at low magnification and in shorter period of time.

Ocular myxoid leiomyosarcoma in a cat.

PubMed

Labelle, Philippe; Holmberg, Bradford J

2010-01-01

A case of myxoid leiomyosarcoma likely of iris dilator muscle origin in the enucleated eye of a 6-year-old domestic short haired cat is reported. The poorly demarcated mass expanded the iris, partially filled the globe and extended into the optic nerve. The mass was composed of spindle cells separated by abundant matrix positive for mucopolysaccharides with alcian blue. The neoplastic cells were immunoreactive for smooth muscle actin (SMA), S100 and vimentin, and negative for cytokeratin, Melan-A, glial fibrillary protein (GFAP) and desmin. There was no evidence of recurrence or metastasis 6 months after enucleation.

Methylene blue intra-arterial staining of resected colorectal cancer specimens improves accuracy of nodal staging: A randomized controlled trial.

PubMed

Reima, H; Saar, H; Innos, K; Soplepmann, J

2016-11-01

Metastatic involvement of regional lymph nodes is a major prognostic factor of colorectal cancer, which influences also its treatment strategy. International consensus foresees retrieval of ≥12 lymph nodes from colorectal specimens. The aim of the study was to assess the effect of intra-arterial staining of colorectal specimens with methylene blue on lymph node harvest. A total of 266 radically operated colorectal cancer patients were randomized into the methylene blue staining and non-staining groups. In the staining group, methylene blue solution was injected into the colorectal specimen's artery after its removal. The specimens were analysed for lymph node count, diameter and metastatic involvement. The median number of lymph nodes was higher in the staining group, 27 (95% CI 23-31%), compared with the control group, 16 (95% CI 14-19, p < 0.001). The number of examined lymph-nodes was ≥12 in 86% of the cases in the staining group and in 69% of the cases in the control group (p = 0.001). In the staining group more small-diameter (≤4 mm) lymph nodes were examined (median number 20.5 vs. 10, p < 0.001). The proportion of patients with metatatic lymph nodes was 42% in the staining group and 43% in the control group (NS). Methylene blue staining improves significantly staging accuracy through finding more small-diameter lymph nodes. It enables to detect ≥12 lymph nodes in the majority of cases. We recommend routine use of this technique in all colorectal resections with curative intent. Copyright © 2016 Elsevier Ltd, BASO ~ the Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Effects of Blue Light Emitting Diode Irradiation On the Proliferation, Apoptosis and Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Yuan, Ye; Yan, Gege; Gong, Rui; Zhang, Lai; Liu, Tianyi; Feng, Chao; Du, Weijie; Wang, Ying; Yang, Fan; Li, Yuan; Guo, Shuyuan; Ding, Fengzhi; Ma, Wenya; Idiiatullina, Elina; Pavlov, Valentin; Han, Zhenbo; Cai, Benzhi; Yang, Lei

2017-01-01

Blue light emitting diodes (LEDs) have been proven to affect the growth of several types of cells. The effects of blue LEDs have not been tested on bone marrow-derived mesenchymal stem cells (BMSCs), which are important for cell-based therapy in various medical fields. Therefore, the aim of this study was to determine the effects of blue LED on the proliferation, apoptosis and osteogenic differentiation of BMSCs. BMSCs were irradiated with a blue LED light at 470 nm for 1 min, 5 min, 10 min, 30 min and 60 min or not irradiated. Cell proliferation was measured by performing cell counting and EdU staining assays. Cell apoptosis was detected by TUNEL staining. Osteogenic differentiation was evaluated by ALP and ARS staining. DCFH-DA staining and γ-H2A.X immunostaining were used to measure intracellular levels of ROS production and DNA damage. Both cell counting and EdU staining assays showed that cell proliferation of BMSCs was significantly reduced upon blue LED irradiation. Furthermore, treatment of BMSCs with LED irradiation was followed by a remarkable increase in apoptosis, indicating that blue LED light induced toxic effects on BMSCs. Likewise, BMSC osteogenic differentiation was inhibited after exposure to blue LED irradiation. Further, blue LED irradiation was followed by the accumulation of ROS production and DNA damage. Taken together, our study demonstrated that blue LED light inhibited cell proliferation, inhibited osteogenic differentiation, and induced apoptosis in BMSCs, which are associated with increased ROS production and DNA damage. These findings may provide important insights for the application of LEDs in future BMSC-based therapies. © 2017 The Author(s). Published by S. Karger AG, Basel.

Brilliant Blue G double staining enhances successful internal limiting membrane peeling with minimal adverse effect by low cellular permeability into live cells.

PubMed

Hisatomi, Toshio; Notomi, Shoji; Tachibana, Takashi; Oishi, Seiichiro; Asato, Ryo; Yamashita, Takehiro; Murakami, Yusuke; Ikeda, Yasuhiro; Enaida, Hiroshi; Sakamoto, Taiji; Ishibashi, Tatsuro

2015-02-01

Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo. A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL. Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained. Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

PubMed

Mizutani, Hitoshi; Akeda, Tomoko; Yamanaka, Kei-Ichi; Isoda, Kenichi; Gabazza, Esteban C

2012-02-01

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist. © 2011 Japanese Dermatological Association.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears.

PubMed

Ratna Kumari, T V N; Ahmed Mujib, B R

2018-01-01

One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears

PubMed Central

Ratna Kumari, T V. N.; Ahmed Mujib, B. R.

2018-01-01

Objectives: One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Study Design: Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. Results: In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Conclusion: Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation. PMID:29403163

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2012 CFR

2012-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2013 CFR

2013-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

Comparison of modified Chicago sky blue stain and potassium hydroxide mount for the diagnosis of dermatomycoses and onychomycoses.

PubMed

Liu, Zhong; Sheng, Ping; Yang, Yan-Ping; Li, Wen; Huang, Wen-Ming; Wang, Jie-Di; Fan, Yi-Ming

2015-05-01

The diagnostic value of modified Chicago sky blue (CSB) stain and potassium hydroxide (KOH) mount for superficial mycoses was compared using fungal culture as gold standard. The sensitivity and screening time of the CSB stain were superior to the KOH mount. The CBS stain is simple, quick and reliable for diagnosing superficial mycoses. Copyright © 2015. Published by Elsevier B.V.

Toluidine blue-O is a Nissl bright-field counterstain for lipophilic fluorescent tracers Di-ASP, DiI and DiO.

PubMed

Chelvanayagam, D K; Beazley, L D

1997-03-01

The stain toluidine blue-O (tol blue), applied to sections of neural tissue, is shown to be compatible with the vivid fluorescent lipophilic neural tracers 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (Di-ASP), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). As with other Nissl stains, toluidine blue-O fluoresces in the red end of the spectrum but such fluorescence quenches upon binding with tissue. Moreover, progressive staining occurs at concentrations low enough to minimise any background fluorescence attributable to non-specific residence of the stain. The bright yellow Di-ASP and vivid green DiO signals are spectrally removed from the red fluorescence of toluidine blue-O. With toluidine blue-O counterstaining, Di-ASP generally offers contrast superior to that with DiI, however, the latter is improved by viewing in a polarised green bright field. Visible Di-ASP emission, although broad, peaks at a more film-sensitive region of the spectrum than that for DiI, thus reducing the photographic exposure required.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

PubMed

Grondin, G; St-Jean, P; Beaudoin, A R

1992-04-01

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

PubMed

Dubový, P; Bednárová, J

1999-12-01

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

Staining paraffin embedded sections of scald of barley before paraffin removal.

PubMed

Xi, K; Burnett, P A

1997-07-01

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and antiline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

PubMed

Raghupathy, V; Oommen, Anna; Ramachandran, Anup

2014-06-15

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

[Localizating and Extracting Small Peripheral Nodules of Lung with Simulating 
Radiaotherapy Combining Methylene Blue Staining].

PubMed

Mao, Feng; Zhang, Liang; Gu, Hengle; Zhang, Hui; Lv, Changxing; Shen-Tu, Yang

2016-09-20

With the extensively application of HRCT (high resolution CT) and the popularization of early lung cancer screening, the proportion of small nodullar lung cancer to be operated increases rapidly. Identifying the focus lesions quickly and accurately in operation has shown to be a challenge. We carried out this research trying to make use of and evaluate a new method that localizaes and extracts small peripheral pulmonary nodules by way of simulating radiaotherapy combining methylene blue staining. From February 2012 to January 2015, 97 patients with 100 peripheral pulmonary nodules ≤10 mm in size were simulated puncturing using a radiotherapy planning. When the anaesthesia came into use, methylene blue dye was injected to the virtually identified point corresponding to the surface point, according to the angle and depth previously computed by the radiotherapy planning. The video-assisted thoracoscopic surgery (VATS) wedge resections of the marked lesions were undertaken and the specimens were sent for frozen pathologic examination. The interval time from anesthesia-completing to puncture and injection, The interval time from methylene blue injection to identifying the stained area and the distances between the centre point of the stains and edge of coloured lesion were recorded. Our preoperative localization procedure was successful in 96 of 100 (96%) nodules. The interval time from anesthesia-completing to puncture and injection of methylene blue were (4.85±1.25) min. The interval time from methylene blue injection to identifying the stained area was (16.36±2.36) min. The distances between the centre point of the stains and edge of coloured lesion were (4.78±2.51) mm. No complication was observed in all participants. The new method of locating peripheral pulmonary nodules by simulating simulating radiaotherapy combining methylene blue staining has a high success rate and no complication for localizing small peripheral pulmonary lesions, avoiding the fear and pain of the patients untaken puncture without anaesthesia reducing radial damage.

Comparison of two modalities: a novel technique, 'chromohysteroscopy', and blind endometrial sampling for the evaluation of abnormal uterine bleeding.

PubMed

Alay, Asli; Usta, Taner A; Ozay, Pinar; Karadugan, Ozgur; Ates, Ugur

2014-05-01

The objective of this study was to compare classical blind endometrial tissue sampling with hysteroscopic biopsy sampling following methylene blue dyeing in premenopausal and postmenopausal patients with abnormal uterine bleeding. A prospective case-control study was carried out in the Office Hysteroscopy Unit. Fifty-four patients with complaints of abnormal uterine bleeding were evaluated. Data of 38 patients were included in the statistical analysis. Three groups were compared by examining samples obtained through hysteroscopic biopsy before and after methylene blue dyeing, and classical blind endometrial tissue sampling. First, uterine cavity was evaluated with office hysteroscopy. Methylene blue dye was administered through the hysteroscopic inlet. Tissue samples were obtained from stained and non-stained areas. Blind endometrial sampling was performed in the same patients immediately after the hysteroscopy procedure. The results of hysteroscopic biopsy from methylene blue stained and non-stained areas and blind biopsy were compared. No statistically significant differences were determined in the comparison of biopsy samples obtained from methylene-blue stained, non-stained areas and blind biopsy (P > 0.05). We suggest that chromohysteroscopy is not superior to endometrial sampling in cases of abnormal uterine bleeding. Further studies with greater sample sizes should be performed to assess the validity of routine use of endometrial dyeing. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.

Computed tomography guided percutaneous injection of a mixture of lipiodol and methylene blue in rabbit lungs: evaluation of localization ability for video-assisted thoracoscopic surgery.

PubMed

Jin, Kwang Nam; Lee, Kyung Won; Kim, Tae Jung; Song, Yong Sub; Kim, Dong Il

2014-01-01

Preoperative localization is necessary prior to video assisted thoracoscopic surgery for the detection of small or deeply located lung nodules. We compared the localization ability of a mixture of lipiodol and methylene blue (MLM) (0.6 mL, 1:5) to methylene blue (0.5 mL) in rabbit lungs. CT-guided percutaneous injections were performed in 21 subjects with MLM and methylene blue. We measured the extent of staining on freshly excised lung and evaluated the subjective localization ability with 4 point scales at 6 and 24 hr after injections. For MLM, radio-opacity was evaluated on the fluoroscopy. We considered score 2 (acceptable) or 3 (excellent) as appropriate for localization. The staining extent of MLM was significantly smaller than methylene blue (0.6 vs 1.0 cm, P<0.001). MLM showed superior staining ability over methylene blue (2.8 vs 2.2, P=0.010). Excellent staining was achieved in 17 subjects (81%) with MLM and 8 (38%) with methylene blue (P=0.011). An acceptable or excellent radio-opacity of MLM was found in 13 subjects (62%). An appropriate localization rate of MLM was 100% with the use of the directly visible ability and radio-opacity of MLM. MLM provides a superior pulmonary localization ability over methylene blue.

Computed Tomography Guided Percutaneous Injection of a Mixture of Lipiodol and Methylene Blue in Rabbit Lungs: Evaluation of Localization Ability for Video-Assisted Thoracoscopic Surgery

PubMed Central

Jin, Kwang Nam; Kim, Tae Jung; Song, Yong Sub; Kim, Dong Il

2014-01-01

Preoperative localization is necessary prior to video assisted thoracoscopic surgery for the detection of small or deeply located lung nodules. We compared the localization ability of a mixture of lipiodol and methylene blue (MLM) (0.6 mL, 1:5) to methylene blue (0.5 mL) in rabbit lungs. CT-guided percutaneous injections were performed in 21 subjects with MLM and methylene blue. We measured the extent of staining on freshly excised lung and evaluated the subjective localization ability with 4 point scales at 6 and 24 hr after injections. For MLM, radio-opacity was evaluated on the fluoroscopy. We considered score 2 (acceptable) or 3 (excellent) as appropriate for localization. The staining extent of MLM was significantly smaller than methylene blue (0.6 vs 1.0 cm, P<0.001). MLM showed superior staining ability over methylene blue (2.8 vs 2.2, P=0.010). Excellent staining was achieved in 17 subjects (81%) with MLM and 8 (38%) with methylene blue (P=0.011). An acceptable or excellent radio-opacity of MLM was found in 13 subjects (62%). An appropriate localization rate of MLM was 100% with the use of the directly visible ability and radio-opacity of MLM. MLM provides a superior pulmonary localization ability over methylene blue. PMID:24431917

Effect of lipiodol and methylene blue on the thoracoscopic preoperative positioning.

PubMed

Zhang, Chuan-Yu; Yu, Hua-Long; Liu, Shi-He; Jiang, Gang; Wang, Yong-Jie

2015-01-01

The aim of this study was to compare and analyze the site-specific accuracy of mixture of lipiodol and methylene blue (MLM) (0.6 ml, 1:5) and pure methylene blue (0.5 ml) on the rabbit lungs. In this study, CT-guided percutaneous injection of MLM and methylene blue. Compare the staining degree by biopsy of lung tissue. Use 4 points system to evaluate the site-specific accuracy at 6h and 24 h after injection. For MLM, evaluate its radiopacity by radiation. When evaluate the positioning, 2 points mean acceptable, 3 points mean excellent. The results indicated that the staining range of MLM is obvious less than that of methylene blue (0.6 vs. 1.0 cm, P<0.01), but the staining capacity of MLM is higher than that of methylene blue (2.8 vs. 2.2, P = 0.01). About the staining abilities which are evaluated as excellent, MLM group accounts for 81%, methylene blue group accounts for 38% (P = 0.011). About the radiopacity which are evaluated as acceptable or excellent, MLM group accounts for 62%. With good direct vision, the suitable positioning rate of MLM can be 100%, which is better than that of methylene blue. In conclusion, percutaneous injection of MLM can be used to lung positioning. The result shows that use MLM is better than only using methylene blue. But it is necessary to do the investigation in human beings in order to confirm the feasibility of its clinical application.

Effect of lipiodol and methylene blue on the thoracoscopic preoperative positioning

PubMed Central

Zhang, Chuan-Yu; Yu, Hua-Long; Liu, Shi-He; Jiang, Gang; Wang, Yong-Jie

2015-01-01

The aim of this study was to compare and analyze the site-specific accuracy of mixture of lipiodol and methylene blue (MLM) (0.6 ml, 1:5) and pure methylene blue (0.5 ml) on the rabbit lungs. In this study, CT-guided percutaneous injection of MLM and methylene blue. Compare the staining degree by biopsy of lung tissue. Use 4 points system to evaluate the site-specific accuracy at 6h and 24 h after injection. For MLM, evaluate its radiopacity by radiation. When evaluate the positioning, 2 points mean acceptable, 3 points mean excellent. The results indicated that the staining range of MLM is obvious less than that of methylene blue (0.6 vs. 1.0 cm, P<0.01), but the staining capacity of MLM is higher than that of methylene blue (2.8 vs. 2.2, P = 0.01). About the staining abilities which are evaluated as excellent, MLM group accounts for 81%, methylene blue group accounts for 38% (P = 0.011). About the radiopacity which are evaluated as acceptable or excellent, MLM group accounts for 62%. With good direct vision, the suitable positioning rate of MLM can be 100%, which is better than that of methylene blue. In conclusion, percutaneous injection of MLM can be used to lung positioning. The result shows that use MLM is better than only using methylene blue. But it is necessary to do the investigation in human beings in order to confirm the feasibility of its clinical application. PMID:26221301

Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach.

PubMed

Magnet, Angela; Gomes, Thiago Santos; Pardinas, Carmen; Garcia de Blas, Natalia; Sadaba, Cruz; Carrillo, Eugenia; Izquierdo, Fernando; Del Castillo, José Manuel Benítez; Hurtado, Carolina; Del Aguila, Carmen; Fenoy, Soledad

2018-01-22

The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Guided surgical debridement: staining tissues with methylene blue.

PubMed

Dorafshar, Amir H; Gitman, Marina; Henry, Ginard; Agarwal, Shailesh; Gottlieb, Lawrence J

2010-01-01

Precise surgical debridement of wounds is required to achieve wound closure. The authors describe their experience with a technique using topical methylene blue to facilitate precise surgical debridement. In this technique, methylene blue dye is applied topically to the wound surface at the onset of surgery. The stained wound site is then wiped to remove dye from the surface of normal epithelium; eschar, nonviable tissue, and granulation tissue remain stained. The methylene blue-stained tissue is surgically removed, and the newly debrided surface of the wound is assessed for adequate vascularity and biopsied to verify presence of bacteriologic balance before closure. The authors have used this technique in more than 200 wound debridements during the past year, including acute surgical or traumatic wounds, acute and subacute burn wounds, chronic granulating wounds, partially epithelialized wounds, sinus tracts, and fistulae. No adverse reactions have been noted, even on patients undergoing multiple applications through serial operations. Topical application of methylene blue to wounds with mixed tissue content helps to distinguish between viable and nonviable tissue and between epithelialized and nonepithelialized areas, facilitating more precise and complete wound debridement.

Genetic diversity of root anatomy in wild and cultivated Manihot species.

PubMed

Bomfim, N N; Graciano-Ribeiro, D; Nassar, N M A

2011-04-05

An anatomical study of roots was conducted on two wild Manihot species, namely M. glaziovii and M. fortalezensis, and two cassava varieties, M. esculenta Crantz variety UnB 201 and M. esculenta variety UnB 122, to identify taxonomic differences in primary growth. Anatomical characters of cassava roots have been rarely investigated. Their study may help cassava breeders to identify varieties with economically important characters, such as tolerance to drought. We investigated tap and lateral adventitious roots of two specimens of each clone or species. Free-hand cross-sections of roots were drawn; these had been clarified with 20% sodium hypochlorite solution, stained with 1% safranin-alcian blue ethanolic solution, dehydrated in ethanol series and butyl acetate and mounted in synthetic resin. Anatomical differences among Manihot species and varieties were found in the epidermal and exodermal cell shape and wall thickness, content of cortical parenchyma, and number of xylem poles. Wall thickness of the epidermis and exodermis of tap root were similar in all species, while in the lateral root there were differences in cell shape and wall thickness. Epidermal cells with thick walls were found in the tap root of all species and in lateral roots of cassava varieties. This character is apparently associated with tolerance to drought and disease. The variation in the number of xylem poles of cassava varieties was larger (4-8) than in wild species (4-6), and appears to support the hybrid origin of cassava.

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

PubMed

Li, Pei; Zhang, Ruijie; Wang, Liyuan; Gan, Yibo; Xu, Yuan; Song, Lei; Luo, Lei; Zhao, Chen; Zhang, Chengmin; Ouyang, Bin; Tu, Bing; Zhou, Qiang

2017-04-30

Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells. © 2017 The Author(s).

Composite poly(l-lactic-acid)/silk fibroin scaffold prepared by electrospinning promotes chondrogenesis for cartilage tissue engineering.

PubMed

Li, Zhengqiang; Liu, Peng; Yang, Ting; Sun, Ying; You, Qi; Li, Jiale; Wang, Zilin; Han, Bing

2016-05-01

Nanofibrous materials produced by electrospinning have attracted considerable attention from researchers in regenerative medicine. A combination of nanofibrous scaffold and chondrocytes is considered promising for repair of cartilage defect or damage. In the present study, we fabricated a poly(l-lactic-acid) (PLLA)/silk fibroin (SF) nanofibrous scaffold by electrospinning and evaluated its chondrogenic potential. The PLLA/SF nanofibers were characterized for diameter, surface wettability, swelling ratio, and tensile strength. Throughin vitroexperiments, PLLA/SF scaffold-chondrocyte interactions were investigated relative to the unmodified PLLA scaffold with regard to cellular adhesion, spreading, and proliferation by scanning electron microscopy and confocal laser scanning microscopy, and through analyses of DNA, sulfated glycosaminoglycan, and collagen. In addition, hematoxylin-eosin and Alcian blue-nuclear fast red staining were used to observe growth of chondrocytes, and secretion and distribution of cartilage-specific extracellular matrices in the scaffolds. Expressions of cartilage-related genes (collagen II, aggrecan, sox9, collagen I, and collagen X) were detected by real-time quantitative PCR. The PLLA/SF scaffold had better hydrophilicity, and could support chondrocytes adhesion and spreading more effectively than the unmodified PLLA scaffold. Chondrocytes secreted more cartilage-specific extracellular matrices and maintained their phenotype on the PLLA/SF scaffold. So it is concluded that the PLLA/SF scaffold is more conducive toin vitroformation of cartilage-like new tissues than the unmodified PLLA scaffold, and may be a promising material in cartilage tissue engineering. © The Author(s) 2016.

Indian and sonic hedgehogs regulate synchondrosis growth plate and cranial base development and function.

PubMed

Young, Blanche; Minugh-Purvis, Nancy; Shimo, Tsuyoshi; St-Jacques, Benoit; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi; Koyama, Eiki; Pacifici, Maurizio

2006-11-01

The synchondroses consist of mirror-image growth plates and are critical for cranial base elongation, but relatively little is known about their formation and regulation. Here we show that synchondrosis development is abnormal in Indian hedgehog-null mice. The Ihh(-/-) cranial bases displayed reduced growth and chondrocyte proliferation, but chondrocyte hypertrophy was widespread. Rather than forming a typical narrow zone, Ihh(-/-) hypertrophic chondrocytes occupied an elongated central portion of each growth plate and were flanked by immature collagen II-expressing chondrocytes facing perichondrial tissues. Endochondral ossification was delayed in much of the Ihh(-/-) cranial bases but, surprisingly, was unaffected most posteriorly. Searching for an explanation, we found that notochord remnants near incipient spheno-occipital synchondroses at E13.5 expressed Sonic hedgehog and local chondrocytes expressed Patched, suggesting that Shh had sustained chondrocyte maturation and occipital ossification. Equally unexpected, Ihh(-/-) growth plates stained poorly with Alcian blue and contained low aggrecan transcript levels. A comparable difference was seen in cultured wild-type versus Ihh(-/-) synchondrosis chondrocytes. Treatment with exogenous Ihh did not fully restore normal proteoglycan levels in mutant cultures, but a combination of Ihh and BMP-2 did. In summary, Ihh is required for multiple processes during synchondrosis and cranial base development, including growth plate zone organization, chondrocyte orientation, and proteoglycan production. The cranial base appears to be a skeletal structure in which growth and ossification patterns along its antero-posterior axis are orchestrated by both Ihh and Shh.

Lubiprostone decreases mouse colonic inner mucus layer thickness and alters intestinal microbiota.

PubMed

Musch, Mark W; Wang, Yunwei; Claud, Erika C; Chang, Eugene B

2013-03-01

Lubiprostone has been used to treat constipation through its effects to stimulate Cl(-) secretion, resulting in water and electrolyte secretion. Potential associated changes in intestinal mucus and the colonizing bacteria (microbiome) have not been studied. As mucus obstructions may play a role in cystic fibrosis, the hypothesis that lubiprostone alters intestinal mucus and the microbiome was investigated. Ion transport studies were performed ex vivo. For mucus and microbiome studies, mice were gavaged daily with lubiprostone or vehicle. Mucin from intestinal sections was analyzed in Carnoy's fixed tissues stained with Alcian blue. Microbiome composition was analyzed by 16S rRNA gene-based sequencing. Lubiprostone stimulated short circuit current in all mouse intestinal segments after both serosal and mucosal additions, albeit at lower concentrations in the latter. Current was Cl-dependent and blocked by mucosal diphenylcarboxylic acid, serosal bumetanide, and serosal Ba(++). The CFTR inhibitor CFTRinh172 had a marginal effect. Mucus near epithelial cells (inner layer mucus) was not present in the small intestine of any mice. Proximal colon inner mucus layer was thicker in ∆F/∆F compared with +/∆F and +/+ mice. Lubiprostone decreased inner mucus layer thickness in both proximal and distal colon of all mice. Furthermore, lubiprostone altered the intestinal microbiome by increasing abundance of Lactobacillus and Alistipes. Lubiprostone activates non-CFTR Cl(-) secretion and alters the colonic inner mucus layer, which is associated with changes in the composition of the enteric microbiome.

Lubiprostone Decreases Mouse Colonic Inner Mucus Layer Thickness and Alters Intestinal Microbiota

PubMed Central

Musch, Mark W.; Wang, Yunwei; Claud, Erika C.

2013-01-01

Background Lubiprostone has been used to treat constipation through its effects to stimulate Cl− secretion, resulting in water and electrolyte secretion. Aim Potential associated changes in intestinal mucus and the colonizing bacteria (microbiome) have not been studied. As mucus obstructions may play a role in cystic fibrosis, the hypothesis that lubiprostone alters intestinal mucus and the microbiome was investigated. Methods Ion transport studies were performed ex vivo. For mucus and microbiome studies, mice were gavaged daily with lubiprostone or vehicle. Mucin from intestinal sections was analyzed in Carnoy’s fixed tissues stained with Alcian blue. Microbiome composition was analyzed by 16S rRNA gene-based sequencing. Results Lubiprostone stimulated short circuit current in all mouse intestinal segments after both serosal and mucosal additions, albeit at lower concentrations in the latter. Current was Cl-dependent and blocked by mucosal diphenylcarboxylic acid, serosal bumetanide, and serosal Ba++. The CFTR inhibitor CFTRinh172 had a marginal effect. Mucus near epithelial cells (inner layer mucus) was not present in the small intestine of any mice. Proximal colon inner mucus layer was thicker in ΔF/ΔF compared with +/ΔF and +/+ mice. Lubiprostone decreased inner mucus layer thickness in both proximal and distal colon of all mice. Furthermore, lubiprostone altered the intestinal microbiome by increasing abundance of Lactobacillus and Alistipes. Conclusions Lubiprostone activates non-CFTR Cl− secretion and alters the colonic inner mucus layer, which is associated with changes in the composition of the enteric microbiome. PMID:23329012

Immunoblotting assays for keratan sulfate.

PubMed

Yoon, Jung Hae; Brooks, Randolph; Halper, Jaroslava

2002-07-15

The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hampered by the lack of sensitive methods. In this paper we describe the modification and development of three sensitive assays capable of detecting nanogram quantities of GAGs in biological samples. The first assay detects total GAGs. It is a modified Alcian blue dye precipitation assay in which the dye binds to the negatively charged GAGs in CsCl-fractionated extracts from chicken tendons. This assay compares favorably with the widely used uronic acid assay in terms of its sensitivity and ability to detect all classes of GAGs, including keratan sulfate (KS). Two other assays, dot-blotting and immunoblotting, detect KS in complex mixtures and can be easily adapted for the detection of other GAGs. Both take advantage of binding of carboxyl and sulfate groups of GAGs to trivalent neodymium. In dot-blotting, samples were directly blotted onto nitrocellulose membrane soaked in Nd(2)(SO(4))(3) buffer, and KS was detected with the monoclonal anti-KS 5-D-4 antibody and an avidin-biotin complex detection system. In immunoblotting, the samples were first separated in 28% polyacrylamide gels, transferred onto a Nd(2)(SO(4))(3)-soaked nitrocellulose membrane using a phosphate buffer system, and stained and developed using the same protocol as in dot-blotting. Whereas dot-blotting allows the use of very low quantities of samples because of its high sensitivity (lower detection limit was 5 ng), immunoblotting provides more specificity.

Barrett’s esophagus and its correlation with gastroesophageal reflux in Chinese

PubMed Central

Zhang, Jun; Chen, Xiao-Li; Wang, Kang-Min; Guo, Xiao-Dan; Zuo, Ai-Li; Gong, Jun

2004-01-01

AIM: To study the prevalence of Barrett’s esophagus in Chinese and its correlation with gastroesophageal reflux. METHODS: This study was carried out in a large prospective series of 391 patients who had undergone upper endoscopy. The patients were divided into 3 groups according to the position of squamocolumnar junction (SCJ). Reflux esophagitis (RE) and its degree were recorded. Intestinal metaplasia (IM) in biopsy specimen was typed according to histochemistry and HE and alcian blue (pH2.5) staining separately. Results correlating with clinical, endoscopic, and pathological data were analysed. RESULTS: The prevalence of IM endoscopically appearing Long-segment Barrett’s Esophagus (LSBE) was 26.53%, Short-segment Barrett’s Esophagus (SSBE) was 33.85% and gastroesophageal junction (GEJ) was 34.00%. IM increased with age of above 40 years old and no difference was found between male and female. Twelve were diagnosed as dysplasia (7 low -grade, 5 high-grade), 16 were diagnosed as cardiac adenocarcinoma and 1 as esophageal adenocarcinoma. The more far away the SCJ moved upward above GEJ, the higher the prevalence and the more severe the RE were. CONCLUSION: There was no difference of the prevalence of IM in different places of SCJ, and IM increased with age of above 40 years old. It is important to pay attention to dysplasia in the distal esophagus and gastro-esophageal junction, and adenocarcinoma is more common in cardia than in esophagus. BE is a consequence of gastroesophageal reflux disease. PMID:15052695

Hyaluronan as a propellant for epithelial movement: the development of semicircular canals in the inner ear of Xenopus.

PubMed

Haddon, C M; Lewis, J H

1991-06-01

The membranous labyrinth of the inner ear, with its three semicircular canals, originates from a simple spheroidal otic vesicle. The process is easily observed in Xenopus. The vesicle develops three dorsal outpocketings; from the two opposite faces of each outpocketing pillars of tissue are protruded into the lumen; and these paired 'axial protrusions' eventually meet and fuse, to form a column of tissue spanning the lumen of the outpocketing like the hub of a wheel, with a tube of epithelium forming the semicircular canal around the periphery. Each axial protrusion consists of epithelium encasing a core of largely cell-free extracellular matrix that stains strongly with alcian blue. In sections, at least 60% of the stainable material is removed by treatment with Streptomyces hyaluronidase. When Streptomyces hyaluronidase is microinjected into the core of a protrusion in vivo, the protrusion collapses and the corresponding semicircular canal fails to form. Hyaluronan (hyaluronic acid) in the core of the protrusion therefore seems to be essential in driving the extension of the protrusion. Autoradiography with tritiated glucosamine indicates that the hyaluronan-rich matrix is synthesised by the epithelium covering the tip of the protrusion; the basal lamina here appears to be discontinuous. These findings indicate that the epithelium of the axial protrusion propels itself into the lumen of the otocyst by localised synthesis of hyaluronan. Hyaluronan may be used in a similar way in the development of other organs, such as the heart and the secondary palate.

Prediction of wood mechanical and chemical properties in the presence and absence of blue stain using two near infrared instruments

Treesearch

Brian K. Via; Chi-Leung So; Todd F. Shupe; Lori G. Eckhardt; Michael Stine; Leslie H. Groom

2005-01-01

The objective of this research was to (a) determine if blue stain in solid wood influenced calibration equations developed from a nonstained wood population, (b) assess the bias introduced when scanning was performed by the slave instrument without calibration transfer from the master instrument and (c) partition absorbance-based variation by instrument, stain and...

Projection Stereolithographic Fabrication of Human Adipose Stem Cell-Incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering.

PubMed

Sun, Aaron X; Lin, Hang; Beck, Angela M; Kilroy, Evan J; Tuan, Rocky S

2015-01-01

The poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL) offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light-based PSL (VL-PSL) system to encapsulate human adipose-derived stem cells (hASCs) into a biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG)]/hyaluronic acid (HA) matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84%) and were uniformly distributed throughout the constructs, which possessed high mechanical properties with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium-treated group (TGF-β3 group), hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 × 10(5) fold increases, respectively compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan-rich extracellular matrix, detected by immunohistochemistry, Alcian blue staining, and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group), cell viability decreased with time (65% at 28 days) and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL and PDLLA-PEG/HA-based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage resurfacing.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures

PubMed Central

Spieker, Janine; Mudersbach, Thomas; Vogel-Höpker, Astrid; Layer, Paul G.

2017-01-01

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation. PMID:28118357

Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

PubMed Central

Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

2005-01-01

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

Not All Lacrimal Epithelial Cells are Created Equal—Heterogeneity of the Rabbit Lacrimal Gland and Differential Secretion

PubMed Central

Ding, Chuanqing; Huang, Jianyan; MacVeigh-Aloni, Michelle; Lu, Michael

2013-01-01

Aims To test the hypotheses that some epithelial cells in the rabbit lacrimal gland (LG) are mucin-secreting cells that are also particularly rich in aquaporin 5 (AQP5) and sodium potassium ATPase β1 subunit (NKAβ1), LG-secreted mucins contribute to the total mucin pool in tear film, and that the rabbit LG is a heterogenic gland where proteins secreted in response to different agonists are varied. Materials and methods LGs were obtained from adult female rabbits and processed for paraffin sections for hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS), mucicarmine, and Alcian blue (pH 0.4, 1.0, and 2.5) for the detection of mucins. Serial sections were used for immunohistochemistry (IHC) and PAS. LG lysates and fluids were assayed by dot blot for detection of mucins, and by SDS-PAGE to detect differences in protein profiles of LG fluids stimulated by different agonists. Results HE staining demonstrated that the LG is a heterogeneous gland where most epithelial cells are serous, while all duct cells are mucous cells. Some acini and individual acinar cells within serous acini are also mucous or seromucous cells and these cells are particularly rich in AQP5 and NKAβ1. Dot blot assay showed the presence of mucins in the LG fluids. The protein profiles of LG fluids from pilocarpine, phenylephrine, and isoproterenol varied significantly, particularly in the mid range. Conclusions Our data indicated that the rabbit LG is a heterogeneous gland that is composed of both serous and mucin-secreting cells, and mucins produced by the LG contribute to the mucin pool in the tear film. The heterogeneity of the rabbit LG supports the notion of differential secretion, i.e. the volume and composition of the LG fluids vary depending on various circumstances in the ocular surface and the body’s needs. PMID:21999223

Methylene Blue as a Diagnostic Aid in the Early Detection of Potentially Malignant and Malignant Lesions of Oral Mucosa.

PubMed

Lejoy, Abraham; Arpita, Rai; Krishna, Burde; Venkatesh, Naikmasur

2016-05-01

In vivo stains are the prompt resources, which have emerged in recent years to aid as clinical diagnostic tools in detecting early potentially malignant and malignant lesions. Toluidine blue, by its property of retaining in the increased DNA and RNA cellular activity areas, aids in delineating the suspicious areas. However, it is hazardous if swallowed, and has been shown to have toxicity to fibroblasts. Methylene blue has a similar chemical structure and exhibits similar physicochemical properties as toluidine blue. It is less toxic to the human body and has recently been proposed for screening some gastrointestinal or prostate tumors. The application of this material in detecting oral lesions has so far not been addressed. The objective of this study was to evaluate the sensitivity and reliability of in vivo staining with methylene blue as a diagnostic adjunct in screening for oral malignant or potentially malignant lesions. The present study involved the examination of 75 patients suspected of having oral malignant or potentially malignant lesions by methylene blue staining. The results of methylene blue uptake were compared with a simultaneous biopsy of these lesions. The overall sensitivity was 95% (100% for malignancy and 92% for potentially malignant lesions) and specificity was 70%. The positive predictive value was 91% and negative predictive value of 80% was observed in the study. We consider that methylene blue staining is a useful diagnostic adjunct in a large, community-based oral cancer screening program for high-risk individuals.

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

PubMed Central

Dzandu, J K; Deh, M E; Barratt, D L; Wise, G E

1984-01-01

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes. Images PMID:6200882

Trypan blue staining of the anterior capsule under an air bubble with a modified cannula.

PubMed

Toprak, Ahmet Baris; Erkin, Esin Fatma; Guler, Cenap

2003-01-01

To attain good visibility of the anterior capsule in the advanced or white cataract, trypan blue 0.1% is used to stain the anterior capsule. The dye is usually injected under an air bubble. However, it is difficult to inject the dye properly due to capillary forces. An ordinary anterior chamber cannula was modified and its coverage area increased to facilitate the staining of the anterior capsule under an air bubble. The anterior capsule was stained properly by using the modified cannula in all of the cases.

Methylene Blue-Guided Debridement as an Intraoperative Adjunct for the Surgical Treatment of Periprosthetic Joint Infection.

PubMed

Shaw, Jeremy D; Miller, Steve; Plourde, Anna; Shaw, Daniel L; Wustrack, Rosanna; Hansen, Erik N

2017-12-01

Current methods to identify infected tissue in periprosthetic joint infection (PJI) are inadequate. The purpose of this study was (1) to assess methylene blue-guided surgical debridement as a novel technique in PJI using quantitative microbiology and (2) to evaluate clinical success based on eradication of infection and infection-free survival. Sixteen total knee arthroplasty patients meeting Musculoskeletal Infection Society criteria for PJI undergoing the first stage of 2-stage exchange arthroplasty were included in this prospective study. Dilute methylene blue (0.1%) was instilled in the knee before debridement, residual dye was removed, and stained tissue was debrided. Paired tissue samples, stained and unstained, were collected from the femur, tibia, and capsule during debridement. Samples were analyzed by neutrophil count, semiquantitative culture, and quantitative polymerase chain reaction (PCR). Clinical success was a secondary outcome. The mean age was 64.0 ± 6.0 years, and follow-up was 24.4 ± 3.5 months. More bacteria were found in methylene blue-stained vs unstained tissue-based on semiquantitative culture (P = .001). PCR for staphylococcal species showed 9-fold greater bioburden in methylene blue-stained vs unstained tissue (P = .02). Tissue pathology found 53 ± 46 polymorphonuclear leukocytes per high-power field in methylene blue-stained vs 4 ± 13 in unstained tissue (P = .0001). All subjects cleared their primary infection and underwent reimplantation. At mean 2-year follow-up, 25% of patients failed secondary to new infection with a different organism. These results suggest a role for methylene blue in providing a visual index of surgical debridement in the treatment of PJI. Copyright © 2017 Elsevier Inc. All rights reserved.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system.

PubMed

Laurinaviciene, Aida; Plancoulaine, Benoit; Baltrusaityte, Indra; Meskauskas, Raimundas; Besusparis, Justinas; Lesciute-Krilaviciene, Daiva; Raudeliunas, Darius; Iqbal, Yasir; Herlin, Paulette; Laurinavicius, Arvydas

2014-01-01

Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system

PubMed Central

2014-01-01

Background Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Methods Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Results Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Conclusions Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools. PMID:25565007

Hematoxylin shortages: their causes and duration, and other dyes that can replace hemalum in routine hematoxylin and eosin staining.

PubMed

Dapson, R; Horobin, R W; Kiernan, J

2010-02-01

The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa.

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

PubMed

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Visualising DNA in Classrooms Using Nile Blue

ERIC Educational Resources Information Center

Milne, Christine; Roche, Scott; McKay, David

2008-01-01

Giving students the opportunity to extract, manipulate and visualise DNA molecules enhances a constructivist approach to learning about modern techniques in biology and biotechnology Visualisation usually requires agarose gel electrophoresis and staining. In this article, we report on an alternative DNA stain, Nile Blue A, that may be used in the…

Cholangiocarcinoma of intrahepatic bile ducts with disseminated metastases in an African lion (Panthera leo).

PubMed

Lepri, Elvio; Sforna, Monica; Brachelente, Chiara; Chiara, Brachelente; Vitellozzi, Giovanni; Giovanni, Vitellozzi

2013-06-01

A cholangiocarcinoma is reported in an 18-yr-old, female African lion (Panthera leo). The primary tumor consisted of multifocal to coalescing, hepatic, white-yellow masses distributed throughout the liver lobes. Metastases were present in regional lymph nodes, peritoneal surface, and lungs. Histologically, the tumor was characterized by a tubular pattern with alcian- and periodic acid-Schiff-positive secretory material in cystic spaces. The neoplastic cells were positive to broad-spectrum cytokeratins. Histochemical and immunohistochemical stains were consistent with bile duct carcinoma. Biliary tumors arising from the gallbladder have been reported in lions. However, to the authors' knowledge, this is the first case of intrahepatic bile duct carcinoma reported in an African lion.

Toluidine Blue 0.05% Vital Staining for the Diagnosis of Ocular Surface Squamous Neoplasia in Kenya.

PubMed

Gichuhi, Stephen; Macharia, Ephantus; Kabiru, Joy; Zindamoyen, Alain M'bongo; Rono, Hilary; Ollando, Ernest; Wanyonyi, Leonard; Wachira, Joseph; Munene, Rhoda; Onyuma, Timothy; Jaoko, Walter G; Sagoo, Mandeep S; Weiss, Helen A; Burton, Matthew J

2015-11-01

Clinical features are unreliable for distinguishing ocular surface squamous neoplasia (OSSN) from benign conjunctival lesions. To evaluate the adverse effects, accuracy, and interobserver variation of toluidine blue 0.05% vital staining in distinguishing OSSN, confirmed by histopathology, from other conjunctival lesions. Cross-sectional study in Kenya from July 2012 through July 2014 of 419 adults with suspicious conjunctival lesions. Pregnant and breastfeeding women were excluded. Comprehensive ophthalmic slitlamp examination was conducted. Vital staining with toluidine blue 0.05% aqueous solution was performed before surgery. Initial safety testing was conducted on large tumors scheduled for exenteration looking for corneal toxicity on histology before testing smaller tumors. We asked about pain or discomfort after staining and evaluated the cornea at the slitlamp for epithelial defects. Lesions were photographed before and after staining. Diagnosis was confirmed by histopathology. Six examiners assessed photographs from a subset of 100 consecutive participants for staining and made a diagnosis of OSSN vs non-OSSN. Staining was compared with histopathology to estimate sensitivity, specificity, and predictive values. Adverse effects were enumerated. Interobserver agreement was estimated using the κ statistic. A total of 143 of 419 participants (34%) had OSSN by histopathology. The median age of all participants was 37 years (interquartile range, 32-45 years) and 278 (66%) were female. A total of 322 of the 419 participants had positive staining while 2 of 419 were equivocal. There was no histological evidence of corneal toxicity. Mild discomfort was reported by 88 (21%) and mild superficial punctate keratopathy seen in 7 (1.7%). For detecting OSSN, toluidine blue had a sensitivity of 92% (95% CI, 87%-96%), specificity of 31% (95% CI, 25%-36%), positive predictive value of 41% (95% CI, 35%-46%), and negative predictive value of 88% (95% CI, 80%-94%). Interobserver agreement was substantial for staining (κ = 0.76) and moderate for diagnosis (κ = 0.40). With the high sensitivity and low specificity for OSSN compared with histopathology among patients with conjunctival lesions, toluidine blue 0.05% vital staining is a good screening tool. However, it is not a good diagnostic tool owing to a high frequency of false-positives. The high negative predictive value suggests that a negative staining result indicates that OSSN is relatively unlikely.

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex.

PubMed

Berggren, K; Chernokalskaya, E; Steinberg, T H; Kemper, C; Lopez, M F; Diwu, Z; Haugland, R P; Patton, W F

2000-07-01

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kawsar, Sarkar M A; Toda, Tosifusa; Kanaly, Robert

2010-11-01

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12ng within 45min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation. 2010 Elsevier Inc. All rights reserved.

Novel methylene blue staining technique for localizing small esophageal leiomyomas during thoracoscopic enucleation.

PubMed

Zhang, Z; Ai, B; Liao, Y; Liu, L; Liu, M

2016-11-01

The treatment of choice for leiomyoma, the most common benign esophageal tumor, is thoracoscopic enucleation. One of the most difficult aspects of thoracoscopic enucleation is the precise localization of small tumors (≤1.5 cm) and tumors without external protrusion. No simple, feasible solutions to this problem are available. We developed a novel methylene blue staining technique to localize small esophageal leiomyomas and evaluated the feasibility of our technique. Between January 2013 and July 2014, eight patients with small esophageal leiomyomas (≤1.5 cm) underwent thoracoscopic enucleation in Tongji Hospital. Preoperative endoscopic ultrasonography was performed in all patients. The leiomyomas were located in the middle (n = 5) and lower (n = 3) thirds of the esophagus. We preoperatively injected 0.5-1.0 mL methylene blue in the submucosa adjacent to the tumors under standard gastroscope guidance. The entire staining process took about 10 minutes. Staining was successful in all patients. The unstained tumor was exposed after the blue-stained mediastinal pleura, and overlying muscle were incised longitudinally. All procedures were successfully completed without conversion to open surgery. No abnormalities were detected in the esophageal mucosa. The median operating time was 60 minutes (range, 40-90 minutes). Postoperative histopathology confirmed leiomyoma in all patients. The median postoperative hospital stay was 6 days (range, 5-7 days). No major complications, such as esophageal leakage or esophageal diverticulum, occurred. Endoscopic methylene blue staining is safe and feasible for localizing small esophageal leiomyomas during thoracoscopic enucleation. This method will enable precise and easy enucleation. © 2015 International Society for Diseases of the Esophagus.

Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

1998-12-01

Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated sialomucins in C9. Adenocarcinomas of the colloid type, thought to bear a poor prognosis, are the most heavily O-acylated.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-05-01

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10 5 (group A) or 8×10 5 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

PubMed

Georgantzopoulou, Anastasia; Serchi, Tommaso; Cambier, Sébastien; Leclercq, Céline C; Renaut, Jenny; Shao, Jia; Kruszewski, Marcin; Lentzen, Esther; Grysan, Patrick; Eswara, Santhana; Audinot, Jean-Nicolas; Contal, Servane; Ziebel, Johanna; Guignard, Cédric; Hoffmann, Lucien; Murk, AlberTinka J; Gutleb, Arno C

2016-02-17

The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and toluidine blue.

PubMed

Epstein, J B; Silverman, S; Epstein, J D; Lonky, S A; Bride, M A

2008-06-01

Conventional visual examination and palpation remains the gold-standard for the identification of oral mucosal lesions. The purpose of this study was to investigate the adjunctive value of a chemiluminescent light source (ViziLite, Zila Pharmaceuticals, Phoenix, Arizona) and application of pharmaceutical grade toluidine blue (TBlue(630), Zila Pharmaceuticals, Phoenix, Arizona) to further assess lesions identified during the conventional oral soft tissue examination. Lesions deemed clinically suspicious by visual examination under incandescent light were further assessed under chemiluminescence and then application of toluidine blue stain. Differences between the conventional visual examination and chemiluminescent examination were noted on four characteristics which may aid in lesion identification. Tissue retention of toluidine blue stain was documented. Each suspicious lesion was biopsied and diagnosed based upon routine histopathology. Both adjunctive exams were evaluated by comparing the histologic diagnosis. The additive value of toluidine blue stain retention was assessed in lesions diagnosed as "serious pathology" defined as severe dysplasia, carcinoma in situ and squamous cell carcinoma. Ninety-seven clinically suspicious lesions in 84 patients were identified. The chemiluminescent exam improved the brightness and/or sharpness of margin in 61.8% of identified lesions. Biopsied lesions with toluidine blue stain retention reduced the false positive rate by 55.26% while maintaining a 100% negative predictive value (NPV). Chemiluminescence was shown to increase the brightness and margins of mucosal lesions in a majority of cases and therefore may assist in identification of mucosal lesions not considered under traditional visual examination. Toluidine blue stain retention was associated with a large reduction in biopsies showing benign histology (false positive biopsy results), while maintaining a 100% NPV for the presence of severe dysplasia or cancer. Practitioners may consider use of these adjuncts in practice, however the results presented are based upon experienced providers in referral centers for mucosal disease or cancer centers and therefore positive findings may be an indication for referral to experienced providers.

Bacterial invasion of the inner ear in association with pneumococcal meningitis.

PubMed

Møller, Martin Nue; Brandt, Christian; Østergaard, Christian; Caye-Thomasen, Per

2014-06-01

To examine the pathways of bacterial invasion and subsequent spreading in the inner ear during pneumococcal meningitis. A well-established adult rat model of Streptococcus pneumoniae meningitis was used. Thirty rats were inoculated intrathecally with S. pneumoniae serotype 1, 3 or 9 V and received no additional treatment. The rats were sacrificed when reaching terminal illness or on Day 7 and then prepared for serial sectioning and PAS-Alcian blue staining for light microscopy. During the first few days after inoculation, bacteria invade the inner ear through the cochlear aqueduct, into the scala tympani of the cochlea (perilymphatic space). From here, bacteria spreads apically toward the helicotrema and subsequently basally through the scala vestibuli, toward the vestibule and the vestibular system. When the bacteria after 5 to 6 days had reached scala vestibuli of the basal turn of the cochlea, hematogenous spreading occurred to the spiral ligament and into the cochlear endolymph, subsequently to the vestibular endolymph. We found no evidence of alternative routes for bacterial invasion in the inner ear. Several internal barriers to bacterial spreading were found within the inner ear. Bacterial elimination was evidenced by engulfment by macrophages within the inner ear. From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment.

Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression.

PubMed

Eo, Seong-Hui; Kim, Song Ja

2017-09-18

Matrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated. In the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E 2 (PGE 2 ) assays, and alcian blue staining to measure sulfated-proteoglycan production. We report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE 2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE 2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13. Results suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.

Unusual mucoepidermoid carcinoma of the liver misdiagnosed as squamous cell carcinoma by intraoperative histological examination

PubMed Central

2014-01-01

As rare condition, mucoepidermoid carcinoma may occur in liver although its etiology and pathogenesis is still unclear. We report here a case of intrahepatic mucoepidermoid carcinoma misdiagnosed as cholangiocarcinoma and squamous cell carcinoma by preoperative radiologic and intraoperative histological examinations, respectively. A 60-year-old woman presented with a 1-month history of progressive jaundice, epigastric discomfort, and weight loss with slightly increased carbohydrate antigen 19-9 (CA19-9). Computed tomography (CT) showed a large tumor, 8.0 cm in diameter, in the left lobe of the liver. A preliminary diagnosis of a cholangiocarcinoma of the liver was made. In the intraoperative histological examination, a diagnosis of squamous cell carcinoma was made based on predominantly invasive epidermoid cells with abundant keratinization and absence of mucin-producing cell component. However, postoperative histological diagnosis of the lesion was mucoepidermiod carcinoma of liver by thoroughly microscopical inspection and the presence of mucin-producing cells confirmed by Alcian blue staining. Despite surgical excision and chemotherapy, the tumor showed very aggressive malignancy with tumor recurrence. The patient died shortly afterward, surviving 6 months after surgery. Due to its rarity and distinct morphological features, mucoepidermoid carcinoma might be erroneously interpreted as squamous cell carcinoma by those who were not familiar with this condition in unusual locations. Therefore, removal of sufficient tissue from different portions of the lesion is essential for the surgeons and pathologists to make a precise diagnosis in the intraoperative histological examination. Virtual slide The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4956311271136060 PMID:24475740

Attenuation of Cigarette Smoke-Induced Airway Mucus Production by Hydrogen-Rich Saline in Rats

PubMed Central

Zhang, Jingxi; Dong, Yuchao; Xu, Wujian; Li, Qiang

2013-01-01

Background Over-production of mucus is an important pathophysiological feature in chronic airway disease such as chronic obstructive pulmonary disease (COPD) and asthma. Cigarette smoking (CS) is the leading cause of COPD. Oxidative stress plays a key role in CS-induced airway abnormal mucus production. Hydrogen protected cells and tissues against oxidative damage by scavenging hydroxyl radicals. In the present study we investigated the effect of hydrogen on CS-induced mucus production in rats. Methods Male Sprague-Dawley rats were divided into four groups: sham control, CS group, hydrogen-rich saline pretreatment group and hydrogen-rich saline control group. Lung morphology and tissue biochemical changes were determined by immunohistochemistry, Alcian Blue/periodic acid-Schiff staining, TUNEL, western blot and realtime RT-PCR. Results Hydrogen-rich saline pretreatment attenuated CS-induced mucus accumulation in the bronchiolar lumen, goblet cell hyperplasia, muc5ac over-expression and abnormal cell apoptosis in the airway epithelium as well as malondialdehyde increase in the BALF. The phosphorylation of EGFR at Tyr1068 and Nrf2 up-regulation expression in the rat lungs challenged by CS exposure were also abrogated by hydrogen-rich saline. Conclusion Hydrogen-rich saline pretreatment ameliorated CS-induced airway mucus production and airway epithelium damage in rats. The protective role of hydrogen on CS-exposed rat lungs was achieved at least partly by its free radical scavenging ability. This is the first report to demonstrate that intraperitoneal administration of hydrogen-rich saline protected rat airways against CS damage and it could be promising in treating abnormal airway mucus production in COPD. PMID:24376700

Resveratrol increases nucleus pulposus matrix synthesis through activating the PI3K/Akt signaling pathway under mechanical compression in a disc organ culture.

PubMed

Han, Xiaorui; Leng, Xiaoming; Zhao, Man; Wu, Mei; Chen, Amei; Hong, Guoju; Sun, Ping

2017-12-22

Disc nucleus pulposus (NP) matrix homeostasis is important for normal disc function. Mechanical overloading seriously decreases matrix synthesis and increases matrix degradation. The present study aims to investigate the effects of resveratrol on disc NP matrix homeostasis under a relatively high-magnitude mechanical compression and the potential mechanism underlying this process. Porcine discs were perfusion-cultured and subjected to a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days in a mechanically active bioreactor. The non-compressed discs were used as controls. Resveratrol was added along with culture medium to observe the effects of resveratrol on NP matrix synthesis under mechanical load respectively. NP matrix synthesis was evaluated by histology, biochemical content (glycosaminoglycan (GAG) and hydroxyproline (HYP)), and expression of matrix macromolecules (aggrecan and collagen II). Results showed that this high-magnitude mechanical compression significantly decreased NP matrix content, indicated by the decreased staining intensity of Alcian Blue and biochemical content (GAG and HYP), and the down-regulated expression of NP matrix macromolecules (aggrecan and collagen II). Further analysis indicated that resveratrol partly stimulated NP matrix synthesis and increased activity of the PI3K/Akt pathway in a dose-dependent manner under mechanical compression. Together, resveratrol is beneficial for disc NP matrix synthesis under mechanical overloading, and the activation of the PI3K/Akt pathway may participate in this regulatory process. Resveratrol may be promising to regenerate mechanical overloading-induced disc degeneration. © 2017 The Author(s).

A novel explanation of corneal clouding in a bone marrow transplant-treated patient with Hurler syndrome

PubMed Central

Yuan, Ching; Bothun, Erick D.; Hardten, David R.; Tolar, Jakub; McLoon, Linda K.

2016-01-01

One common complication of mucopolysaccharidosis I-Hurler (MPS1-H) is corneal clouding, which occurs despite current treatments, including bone marrow transplantation. Human corneas were obtained from a 14 year old subject with MPS1-H and visual disability from progressive corneal clouding despite a prior bone marrow transplant at age 2. This was compared to a cornea from a 17 year old donated to our eye bank after his accidental death. The corneas were analyzed microscopically after staining with Alcian blue, antibodies to collagen I, IV, VI, and α-smooth muscle actin. Differences in levels of expression of the indicated molecules were assessed. Corneas from Hurler and control mice were examined similarly to determine potential mechanistic overlap. The MPS1-H subject cornea showed elevations in mucopolysaccharide deposition. The MPS1-H and Hurler mice corneas showed increased and disorganized expression of collagen I and IV relative to the control corneas. The MPS1-H corneas also showed increased and disordered expression of collagen VI. Positive expression of α-smooth muscle actin indicated myofibroblast conversion within the MPS1-H cornea in both the patient and mutant mouse material compared to normal human and control mouse cornea. Increased deposition of collagens and smooth muscle actin correlate with corneal clouding, providing a potential mechanism for corneal clouding despite bone marrow transplantation in MPS1-H patients. It might be possible to prevent or slow the onset of corneal clouding by treating the cornea with drugs known to prevent myofibroblast conversion. PMID:27235795

Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms

PubMed Central

Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G.

2016-01-01

Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein. PMID:27574787

Spiral ganglion degeneration and hearing loss as a consequence of satellite cell death in saposin B-deficient mice.

PubMed

Akil, Omar; Sun, Ying; Vijayakumar, Sarath; Zhang, Wujuan; Ku, Tiffany; Lee, Chi-Kyou; Jones, Sherri; Grabowski, Gregory A; Lustig, Lawrence R

2015-02-18

Saposin B (Sap B) is an essential activator protein for arylsulfatase A in the hydrolysis of sulfatide, a lipid component of myelin. To study Sap B's role in hearing and balance, a Sap B-deficient (B(-/-)) mouse was evaluated. At both light and electron microscopy (EM) levels, inclusion body accumulation was seen in satellite cells surrounding spiral ganglion (SG) neurons from postnatal month 1 onward, progressing into large vacuoles preceding satellite cell degeneration, and followed by SG degeneration. EM also revealed reduced or absent myelin sheaths in SG neurons from postnatal month 8 onwards. Hearing loss was initially seen at postnatal month 6 and progressed thereafter for frequency-specific stimuli, whereas click responses became abnormal from postnatal month 13 onward. The progressive hearing loss correlated with the accumulation of inclusion bodies in the satellite cells and their subsequent degeneration. Outer hair cell numbers and efferent function measures (distortion product otoacoustic emissions and contralateral suppression) were normal in the B(-/-) mice throughout this period. Alcian blue staining of SGs demonstrated that these inclusion bodies corresponded to sulfatide accumulation. In contrast, changes in the vestibular system were much milder, but caused severe physiologic deficits. These results demonstrate that loss of Sap B function leads to progressive sulfatide accumulation in satellite cells surrounding the SG neurons, leading to satellite cell degeneration and subsequent SG degeneration with a resultant loss of hearing. Relative sparing of the efferent auditory and vestibular neurons suggests that alternate glycosphingolipid metabolic pathways predominate in these other systems. Copyright © 2015 the authors 0270-6474/15/353263-13$15.00/0.

Gelatinous Marrow Transformation: A Series of 11 Cases from a Tertiary Care Centre in South India

PubMed Central

Das, Sreeya; Mishra, Pritinanda; Kar, Rakhee; Basu, Debdatta

2014-01-01

Gelatinous marrow transformation (GMT) or serous atrophy of bone marrow (BM) is a rare disease characterised by focal marrow hypoplasia, fat atrophy, and accumulation of extracellular mucopolysaccharides abundant in hyaluronic acid. This study reviews 11 cases of GMT from South India. Clinical and haematological parameters, BM aspirate, and biopsies of all patients diagnosed with GMT over a period of 7 years were studied. GMT was diagnosed in BM biopsy based on characteristic morphological appearance and was confirmed by alcian blue positive staining pattern at pH levels of 2.5 and 0.5. Eleven patients were diagnosed with GMT. All were males within the age range of 15 to 50 years. The underlying clinical diagnosis was human immunodeficiency virus positivity in 5 cases, 2 with coexistent disseminated tuberculosis, 1 with cryptococcal meningitis, and 1 with oral candidiasis; disseminated tuberculosis in 1 case; pyrexia of unknown origin in 2 cases; Hodgkin’s lymphoma in 1 case; acute lymphoblastic lymphoma with maintenance chemotherapy in 1 case; and alcoholic pancreatitis in 1 case. BM aspirates showed gelatinous metachromatic seromucinous material in 3 cases. BM biopsies were hypocellular in 7 and normocellular in 4 cases and showed focal GMT in 5 and diffuse GMT in 6 cases. Reactive changes were seen in 4 cases and haemophagocytosis in addition to GMT in 1 case. GMT is a relatively uncommon condition and an indicator of severe illness. It should be differentiated from myelonecrosis, amyloidosis, and marrow oedema. A high index of suspicion is required to diagnose this condition. PMID:25035676

Gelatinous marrow transformation: a series of 11 cases from a tertiary care centre in South India.

PubMed

Das, Sreeya; Mishra, Pritinanda; Kar, Rakhee; Basu, Debdatta

2014-06-01

Gelatinous marrow transformation (GMT) or serous atrophy of bone marrow (BM) is a rare disease characterised by focal marrow hypoplasia, fat atrophy, and accumulation of extracellular mucopolysaccharides abundant in hyaluronic acid. This study reviews 11 cases of GMT from South India. Clinical and haematological parameters, BM aspirate, and biopsies of all patients diagnosed with GMT over a period of 7 years were studied. GMT was diagnosed in BM biopsy based on characteristic morphological appearance and was confirmed by alcian blue positive staining pattern at pH levels of 2.5 and 0.5. Eleven patients were diagnosed with GMT. All were males within the age range of 15 to 50 years. The underlying clinical diagnosis was human immunodeficiency virus positivity in 5 cases, 2 with coexistent disseminated tuberculosis, 1 with cryptococcal meningitis, and 1 with oral candidiasis; disseminated tuberculosis in 1 case; pyrexia of unknown origin in 2 cases; Hodgkin's lymphoma in 1 case; acute lymphoblastic lymphoma with maintenance chemotherapy in 1 case; and alcoholic pancreatitis in 1 case. BM aspirates showed gelatinous metachromatic seromucinous material in 3 cases. BM biopsies were hypocellular in 7 and normocellular in 4 cases and showed focal GMT in 5 and diffuse GMT in 6 cases. Reactive changes were seen in 4 cases and haemophagocytosis in addition to GMT in 1 case. GMT is a relatively uncommon condition and an indicator of severe illness. It should be differentiated from myelonecrosis, amyloidosis, and marrow oedema. A high index of suspicion is required to diagnose this condition.

Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms.

PubMed

Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G

2016-01-01

Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein.

Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone(r)) osteogenesis: a study on biopsies from human jaws.

PubMed

Götz, Werner; Gerber, Thomas; Michel, Barbara; Lossdörfer, Stefan; Henkel, Kai-Olaf; Heinemann, Friedhelm

2008-10-01

Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the host's physiological bone turnover at a very early stage.

A case of radiation-induced generalized morphea with prominent mucin deposition and tenderness.

PubMed

Yanaba, Koichi; Umezawa, Yoshinori; Nakagawa, Hidemi

2015-05-10

Radiation-induced morphea is a rare complication of radiation therapy. The affected areas are generally restricted to the radiation field or to the nearby surrounding area. A 67-year-old Japanese woman with a history of right breast cancer followed by adjuvant radiotherapy was referred our hospital because of 7-year history of symmetrical indurated erythematous plaques on her trunk. Three months after completion of irradiation, erythematous plaques developed on her right chest and gradually spread accompanied tenderness. She did not have a history of trauma to her right chest. Laboratory testing was positive for antinuclear antibody test at 1: 640 but negative for anti-SS-A/B, anti-U1-RNP, anti-DNA, anti-Sm, anticentromere, anti-topoisomerase I antibodies, and Borrelia and cytomegalovirus infection. She had no Raynaud's phenomenon, sclerodactyly, or nail-fold bleeding. She did not have interstitial lung disease or other internal organ involvement. A biopsy specimen revealed reticular dermal fibrosis with thickened collagen bundles with superficial and deep perivascular infiltration of mononuclear cells. These findings were consistent with morphea. Furthermore, mucin deposition was present in the papillary dermis upon Alcian blue staining, which has been reported to be observed in generalized morphea. Consequently, a diagnosis of generalized morphea induced by radiotherapy was made. She had been treated with oral hydroxychloroquine sulfate, resulting in the resolution of tenderness but the erythematous plaques remained. To the best of our knowledge, this is the first report of radiation-induced generalized morphea with prominent mucin deposition. Hydroxychloroquine sulfate may be efficacious for radiation-induced morphea-associated tenderness.

Chondrogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Chitosan Film Culture

PubMed Central

Lu, Tsai-Jung; Chiu, Fang-Yao; Chiu, Hsiao-Ying; Chang, Ming-Chau; Hung, Shih-Chieh

2017-01-01

Articular cartilage has a very limited capacity for self-repair, and mesenchymal stem cells (MSCs) have the potential to treat cartilage defects and osteoarthritis. However, in-depth mechanistic studies regarding their applications are required. Here we demonstrated the use of chitosan film culture for promoting chondrogenic differentiation of MSCs. We found that MSCs formed spheres 2 days after seeding on dishes coated with chitosan. When MSCs were induced in a chondrogenic induction medium on chitosan films, the size of the spheres continuously increased for up to 21 days. Alcian blue staining and immunohistochemistry demonstrated the expression of chondrogenic proteins, including aggrecan, type II collagen, and type X collagen at 14 and 21 days of differentiation. Importantly, chitosan, with a medium molecular weight (size: 190–310 kDa), was more suitable than other sizes for inducing chondrogenic differentiation of MSCs in terms of sphere size and expression of chondrogenic proteins and endochondral markers. We identified that the mechanistic target of rapamycin (mTOR) signaling and its downstream S6 kinase (S6K)/S6 were activated in chitosan film culture compared to that of monolayer culture. The activation of mTOR/S6K was continuously upregulated from days 2 to 7 of differentiation. Furthermore, we found that mTOR/S6K signaling was required for chondrogenic differentiation of MSCs in chitosan film culture through rapamycin treatment and mTOR knockdown. In conclusion, we showed the suitability of chitosan film culture for promoting chondrogenic differentiation of MSCs and its potential in the development of new strategies in cartilage tissue engineering. PMID:27737727

Control of brown stain: in Eastern white pine

Treesearch

Robert E. Stutz; Peter Koch; Millard L. Oldham

1961-01-01

Degrade caused by brown stain and blue stain in eastern white pine was virtually eliminated by the use of sap stain chemicals and sodium azide. Combinations of buffered sodium azide with both sodium pentachlorophenate plus borax and buffered ethyl mercury phosphate were effective.

Assessment of oral cytological features in smokers and nonsmokers after application of toluidine blue.

PubMed

Sharbatdaran, Majid; Abbaszadeh, Hamid; Siadati, Sepideh; Ranaee, Mohammad; Hajian-Tilaki, Karimollah; Rajabi-Moghaddam, Mahdieh

2017-06-01

Smoking is the most important etiologic factor of oral cancer. Exfoliative cytology is the best method for early detection of oral cancer. Toluidine blue staining is used for detection of oral premalignant and malignant lesions. The aim of this study was to enhance the accuracy of oral exfoliative cytology in evaluating dysplastic features using toluidine blue staining. This clinical trials study was performed on 60 male smokers and nonsmokers without clinically oral lesion. Oral exfoliative cytological smears were prepared before and after application of toluidine blue and stained with Papanicolaou and evaluated under light microscope. Cytological features such as cellular clumping nuclear-to-cytoplasmic ratio, cellular and nuclear pleomorphism, micronuclei, binucleation, presence of bacterial colonies, and keratin flakes were assessed and compared before and after application of toluidine blue. Results showed that cellular clumping and micronuclei were significantly decreased after application of toluidine blue and conversely cellular and nuclear pleomorphisms were significantly increased. Frequency of micronuclei and binucleation were greater in smokers than nonsmokers which were insignificant. Cellular and nuclear pleomorphisms were significantly higher in smokers than nonsmokers after application of toluidine blue. Toluidine blue improved cellular, nuclear, and structural features of oral cytological smears and filtered false-positive or false-negative results. Thus, application of toluidine blue in combination with oral exfoliative cytology for early detection of oral cancer is recommended. Diagn. Cytopathol. 2017;45:513-519. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A comparative analysis of toluidine blue with frozen section in oral squamous cell carcinoma

PubMed Central

2012-01-01

Background Surgical excision of the primary tumor with safe margins remains the mainstay of treatment for oral cavity squamous cell carcinoma (OSCC). The standard of care for assessment of intraoperative margins is frozen section histopathology. Unfortunately the facility is not available at most centers in limited resource countries. Toluidine blue, a metachromatic dye, has been well described in clinical identification of malignant and premalignant lesion in the oral cavity. Considering this we decided to explore intraoperative use of toluidine blue staining, in comparison with frozen sections, for the assessment of tumor-free margins. Methods After obtaining clearance from the in-house ethical review committee, a prospective study was conducted at Aga Khan University Hospital, Karachi, from August 15, 2009 to March 14, 2010. A sample of 56 consenting patients with biopsy-proven OSCC were included in the study, giving us 280 tumor margins. Margins were analyzed using toluidine blue staining and frozen section histopathology. A receiver operator curve (ROC) was then applied to compare assessment of margin status by toluidine blue and frozen section. Results Of the 280 examined margins 11 stained positive with toluidine blue, three were positive on frozen section biopsy, and three were positive on final histopathology. Toluidine blue staining had sensitivity and specificity of 100% and 97%, respectively. The diagnostic accuracy of toluidine blue was found to be 97.1% with a positive predictive value (PPV) of 27.2% and a negative predictive value (NPV) of 100%. Conclusions Toluidine blue can be used as an effective screening modality for the assessment of intraoperative margins in resource limited environments and reducing the number of frozen section biopsies performed. Further by providing real-time clinical information within minutes it can reduce indirect costs such as operating room time. It may also be used as an ad hoc for frozen section biopsies where frozen section facilities are available. PMID:22500814

Influence of hypodermic needle dimensions on subcutaneous injection delivery--a pig study of injection deposition evaluated by CT scanning, histology, and backflow.

PubMed

Juul, Kezia Ann Praestmark; Bengtsson, Henrik; Eyving, Bente; Kildegaard, Jonas; Lav, Steffen; Poulsen, Mette; Serup, Jørgen; Stallknecht, Bente

2012-11-01

Thinner and shorter needles for subcutaneous administration are continuously developed. Previous studies have shown that a thinner needle causes fewer occurrences of painful needle insertions and that a shorter needle decreases the occurrence of painful intramuscular injections. However, little is known about local drug delivery in relation to needle length and thickness. This study aimed to compare deposition depth and backflow from three hypodermic needles of 3 mm 34G (0.19 mm), 5 mm 32G (0.23 mm), and 8 mm 30G (0.30 mm) in length and thickness. Ex vivo experiments were carried out on pigs, in neck tissue comparable to human skin at typical injection sites. Six pigs were included and a total of 72 randomized injections were given, i.e. 24 subcutaneous injections given with each needle type. Accordingly, 400 μL was injected including 70% NovoRapid(®) (Novo Nordisk A/S, Bagsvμrd, Denmark) and 30% Xenetix(®) (Guerbet, Villepinte, France) contrast including 1 mg/mL Alcian blue. Surgical biopsies of injection sites were sampled and computer topographic (CT)-scanned in 3D to assess deposition and local distribution. Biopsies were prepared and stained to evaluate deposition in comparison to the CT-scanning findings. The backflow of each injection was collected with filter paper. The blue stains of filter paper were digitized and volume estimated by software calculation vs. control staining. CT-scanning (n = 57) and histology (n = 10) showed that, regardless of injection depth, the bulk of the injection was in the subcutaneous tissue and did not propagate from subcutis into dermis. With the 8 mm 30G needle all injections apart from one intramuscular injection were located in the subcutaneous layer. The volume depositions peaked in 4-5 mm depth for the 3 mm 34G needle, in 5-6 mm depth for the 5 mm 32G needle, and in 9-10 mm depth for the 8 mm 30G needle. In general, injection depositions evaluated by histology and CT-scans compared well for the individual biopsies. The amount of backflow (n = 69) from the 3 mm 34G needle was smaller (P < 0.05) as compared to the 5 mm 32G and the 8 mm 30G needles. Analysis showed a correlation between backflow and the needle's outer dimension with the needle diameter being the pivotal parameter. Furthermore, CT-scanning and histology confirmed that local propagation of the injection and final distribution followed a route of less mechanical resistance as determined by connective tissue barriers preset in the site of injection. Needles as short as 3 mm efficiently delivered injections into the subcutaneous target. The amount of backflow was smaller with thinner needles. Local distribution was variable and determined by mechanical barriers preset in the tissue. CT-scans and histology were concordant. © 2012 John Wiley & Sons A/S.

Response of Lutz, Sitka, and white spruce to attack by Dendroctonus rufipennis (Coleoptera: Scolytidae) and blue stain fungi

Treesearch

Richard A. Werner; Barbara L. Illman

1994-01-01

Mechanical wounding and wounding plus inoculation with a blue-stain fungus, Leptographium abietinum (Peck), associated with the spruce beetle, Dendroctonus rufipennis (Kirby), caused an induced reaction zone or lesion around the wound sites in Lutz spruce, Picea lutzii Little, Sitka spruce, P. sitchensis (Bong.) Carr., and white spruce, P. glauca (Moench) Voss, in...

Evaluation of white-rot fungal growth on southern yellow pine wood chips pretreated with blue-stain fungi

Treesearch

Suki C. Croan

2000-01-01

White-rotting basidiomycetes do not colonize on southern yellow pine. This study seeks to reduce the resinous extractive content of southern yellow pine by treating it with blue stain fungi. The mycelial growth of wood-inhabiting ligninolytic white-rot fungi can be achieved on pretreated southern yellow pine wood. Aureobasidium, Ceratocystis, and Ophiostoma spp....

Quirks of dye nomenclature. 8. Methylene blue, azure and violet.

PubMed

Cooksey, C J

2017-01-01

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.

The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution

NASA Astrophysics Data System (ADS)

Oktari, A.; Supriatin, Y.; Kamal, M.; Syafrullah, H.

2017-02-01

Endospores staining is the type of staining to recognize the presence spore in bacterial vegetative cells. The bacterial endospores need a staining which can penetrate wall thickness of spore bacteria. A method of endospores staining is Schaeffer Fulton method that used Malachite Green. It is an alkaline substance staining that can staining the spore bacteria. In this research, it have found the alternative staining that can replace Malachite Green solution in spore bacterial stain. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%, and 1% concentration) with pH variation (10, 11, and 12), and varyous heating time (3, 4, and 5 minutes). The all treatments staining have been effect on bacterial spores staining results. The warming time greatly affect the dye to penetrate the walls of bacterial spores, this can be seen in the results with various concentration at pH 10, indicates that the not long warm-up time 3 and 4 minutes, bacterial spores are not stained, while in the longer heating time is 5 minutes bacterial spores stained. This is caused because the longer heating time can make the pores of spore wall is open so that can facilitate the dye to get into the bacterial spores.

Blue-stain Fungi Associated with Roots of Southern Pine Trees Attacked by the Southern Pine Beetle, Dendroctonus frontalis

Treesearch

William J. Otrosina; Nolan J. Hess; Stanley J. Zarnoch; Thelma J. Perry; John P. Jones

1997-01-01

Forty paired plots were established from eastern Texas to Alabama to study root-infecting, blue-stain fungi in southern pine stands undergoing southern pine beetle (SPB) attack. Woody roots were sampled in plots undergoing recent or current attack by the SPB. Comparisons were made between occurrence of Lcptogrqhiumspp. and related fungi and data on various...

Gastroprotective Activity of Ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylidene) Amino]benzoate against Ethanol-Induced Gastric Mucosal Ulcer in Rats

PubMed Central

Halabi, Mohammed Farouq; Shakir, Raied Mustafa; Bardi, Daleya Abdulaziz; Al-Wajeeh, Nahla Saeed; Ablat, Abdulwali; Hassandarvish, Pouya; Hajrezaie, Maryam; Norazit, Anwar; Abdulla, Mahmood Ameen

2014-01-01

Background The study was carried out to determine the cytotoxic, antioxidant and gastro-protective effect of ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylid ene)amino] benzoate (ETHAB) in rats. Methodology/Principal Findings The cytotoxic effect of ETHAB was assessed using a MTT cleavage assay on a WRL68 cell line, while its antioxidant activity was evaluated in vitro. In the anti-ulcer study, rats were divided into six groups. Group 1 and group 2 received 10% Tween 20 (vehicle). Group 3 received 20 mg/kg Omeprazole. Groups 4, 5 and 6 received ETHAB at doses of 5, 10, and 20 mg/kg, respectively. After an hour, group 1 received the vehicle. Groups 2–6 received absolute ethanol to induce gastric mucosal lesions. In the WRL68 cell line, an IC50 of more than 100 µg/mL was observed. ETHAB results showed antioxidant activity in the DPPH, FRAP, nitric oxide and metal chelating assays. There was no acute toxicity even at the highest dosage (1000 mg/kg). Microscopy showed that rats pretreated with ETHAB revealed protection of gastric mucosa as ascertained by significant increases in superoxide dismutase (SOD), pH level, mucus secretion, reduced gastric lesions, malondialdehyde (MDA) level and remarkable flattened gastric mucosa. Histologically, pretreatment with ETHAB resulted in comparatively better gastric protection, due to reduction of submucosal edema with leucocyte infiltration. PAS staining showed increased intensity in uptake of Alcian blue. In terms of immunohistochemistry, ETHAB showed down-expression of Bax proteins and over-expression of Hsp70 proteins. Conclusion/Significance The gastroprotective effect of ETHAB may be attributed to antioxidant activity, increased gastric wall mucus, pH level of gastric contents, SOD activity, decrease in MDA level, ulcer area, flattening of gastric mucosa, reduction of edema and leucocyte infiltration of the submucosal layer, increased PAS staining, up-regulation of Hsp70 protein and suppressed expression of Bax. Key words: ethyl 4-(3, 5-di-ter-butyl-2-hydroxybenzylamino) benzoate; toxicity; antioxidant; gastric-ulcer; anti-ulcer; histology; immunohistochemistry. PMID:24800807

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution.

PubMed

Steinberg, T H; Lauber, W M; Berggren, K; Kemper, C; Yue, S; Patton, W F

2000-02-01

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture

PubMed Central

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

Background: The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. Aims and Objectives: This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Materials and Methods: Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Observations and Results: Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). Conclusion: CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount. PMID:26288400

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture.

PubMed

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount.

Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases.

PubMed

Gómez Ramírez, M; Rojas Avelizapa, L I; Rojas Avelizapa, N G; Cruz Camarillo, R

2004-02-01

A simple and sensitive method based on the use of colloidal chitin stained with Remazol Brilliant Blue R (RBB) is proposed to evaluate chitinase activity. If this colloidal-stained substrate is included as a carbon source in a liquid medium, this technique allows the selection or the comparison of chitinolytic microorganisms. The colloidal substrate is proportionally solubilized and the dye released is spectrophotometrically quantified at 595 nm. The procedures used for the staining and fixing of RBB in the colloidal chitin, and a comparison with the commercial substrate chitin-azure, are presented. The influence of several physicochemical and enzymatic parameters on the release of dyes is also shown. Both stained substrates were used for studying the effect of pH, substrate concentration, temperature and time on the chitinase reaction of Bacillus thuringiensis Bt-107.

Methylene Blue: The Long and Winding Road from Stain to Brain: Part 1.

PubMed

Howland, Robert H

2016-09-01

Methylene blue, first discovered and used as a dye in the textile industry, has long been used for biological staining in histology, bacteriology, and hematology. Because of its unique physiochemical properties, it was the first synthetic drug used in medicine, having been used to treat malaria more than one century ago. Methylene blue was also one of the first drugs used for the treatment of patients with psychosis at the end of the 19th century and was the lead drug in the serendipitous development of phenothiazine antipsychotic drugs in the mid-20th century. It was studied in bipolar disorder in the 1980s and has been investigated in neurodegenerative disorders in recent years. The history of methylene blue from its discovery as a dye to its use as a stain and then its therapeutic application in medicine is an example of how a drug's use can evolve over time through careful observation, clinical needs, serendipity, and the integration of concepts from different disciplines. [Journal of Psychosocial Nursing and Mental Health Services, 54(9), 21-24.]. Copyright 2016, SLACK Incorporated.

Cement line staining in undecalcified thin sections of cortical bone

NASA Technical Reports Server (NTRS)

Bain, S. D.; Impeduglia, T. M.; Rubin, C. T.

1990-01-01

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

Evaluation of gram stain as an alternative in the assessment of human spermatozoa quality.

PubMed

Mantas, D; Msaouel, P; Angelopoulou, R

2006-01-01

During spermiogenesis, protaminosis and sperm chromatin condensation are important prerequisites for the preservation of DNA integrity in spermatozoa. The aim of this study is to assess Gram stain as an alternative technique for the evaluation of human sperm chromatin condensation status. Aniline blue and Gram staining were applied to semen samples from 34 donors in order to determine the relationship between sperm chromatin condensation and infertility. In addition, the possible correlation between morphology and vitality (eosin-Y staining) of spermatozoa compared with their nuclear status (aniline blue and Gram staining) was studied. Chromatin condensation and sperm vitality were significantly higher in fertile men compared to the subfertile. A significant correlation was found between chromatin condensation and (a) sperm vitality (p < 0.01), and (b) nuclear protein status (p < 0.01). Gram staining may be used as a routine method in assisted reproduction laboratories and could assist in the evaluation of sperm quality as well as in the selection of the appropriate fertilization technique.

Adult Rat Bones Maintain Distinct Regionalized Expression of Markers Associated with Their Development

PubMed Central

Rawlinson, Simon C. F.; McKay, Ian J.; Ghuman, Mandeep; Wellmann, Claudia; Ryan, Paul; Prajaneh, Saengsome; Zaman, Gul; Hughes, Francis J.; Kingsmill, Virginia J.

2009-01-01

The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed. PMID:20027296

Chronic gastritis with intestinal metaplasia: clinico-statistical, histological and immunohistochemical study.

PubMed

Dîrnu, Rodica; Secureanu, F A; Neamţu, Carmen; Totolici, B D; Pop, O T; Mitruţ, P; Mălăescu, D Gh; Mogoantă, L

2012-01-01

Chronic gastritis has a high incidence in adults, causing progressive destruction of glandular structures, favoring the development of gastric atrophy. The association of chronic gastritis with intestinal type metaplasia of gastric mucosa has a poor outcome as intestinal metaplasia is regarded as a precancerous lesion. Metaplasia is common in patients with Helicobacter pylori infection and also heavy smokers. The aim of our study was to evaluate the relationship between chronic gastritis and intestinal metaplasia. The study was conducted on a total of 1218 patients, aged between 5 and 90 years, who presented for dyspeptic disorders in the period 2007-2010 and were examined clinically and endoscopically. During the gastroscopic examination, fragments of gastric mucosa were collected for the histopathological study and for highlighting the H. pylori infection. For the histopathological study, the Hematoxylin-Eosin and PAS-Alcian Blue stains were performed, while for the immunohistochemical study the anti-TAG72 and anti-PCNA antibodies were used. A diagnosis of gastritis was established in 615 patients, representing approximately 50.5% of all cases. Most cases with gastritis were found in people of middle age. Gastritis was present in almost all age groups, from teenagers to the elders. Of the 615 cases of gastritis, urease test was positive in 353 patients, representing approximately 57.40% of all patients with gastritis. Histopathological examination identified the presence of intestinal metaplasia in 61.60% of patients with chronic gastritis, mostly complete metaplasia. PCNA immunohistochemistry revealed that cell proliferation processes are intensified in intestinal metaplasia. This study highlights the importance of chronic gastritis, intestinal metaplasia, and H. pylori infection in the etiopathogeny of gastric cancer.

A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

PubMed Central

López-Igual, Rocío; Lechno-Yossef, Sigal; Fan, Qing; Herrero, Antonia; Wolk, C. Peter

2012-01-01

Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O2) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox− phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox− mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the PglnA promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep. PMID:22753066

Ki-67 and MCM-2 in Dental Follicle and Odontogenic Cysts: The Effects of Inflammation on Proliferative Markers

PubMed Central

Güler, Nurhan; Çomunoğlu, Nil; Cabbar, Fatih

2012-01-01

The aim of this study was to investigate whether there is any association between inflammation and the expression of markers of cell cycle entry (Ki-67 and MCM-2) in dental follicle (DF) of asymptomatic impacted teeth and odontogenic cysts. The study consisted of 70 DFs and 20 odontogenic cysts (radicular cyst (RC), dentigerous cyst (DC) and keratocytic odontogenic tumor (KCOT) located at posterior mandibular region. Histological findings of inflammation for all specimen and mucous cell prosoplasia, squamous metaplasia, glandular epithelium for all DFs were stained with hematoxyline and eosin, periodic acid schiff, alcian blue, and mucin. Epithelial cell proliferation was determined by using immunohistochemical labeling for Ki-67 and MCM-2. The histologic examinations showed 16% mucous cell prosoplasia, 54% squamous metaplasia, 20% glandular epithelium, 37% inflammation. Inflammation was detected in all RCs and %62 in DF, %43 in DC and KCOT. Positive correlation was found between the inflammation of DF and odontogenic cysts (P < 0.01). The mean Ki-67 and MCM-2 expressions were found 9, 64 ± 5, 99 and 6, 34 ± 3, 81 in DF, 11, 85 ± 9, 01 and 13, 6 ± 9, 94 in odontogenic cysts, respectively. While the mean Ki-67 expressions were statistically significant in DF and KCOT (P < 0.01), MCM-2 were significant in RC and KCOT (P < 0.01). MCM-2 expresion in RCs were statistically significant than KCOT (P < 0.01). The results of this study indicated that the higher MCM-2 expressions in RC than the KCOT might be related to the inflammation and this protein might be more sensitive to inflammation. PMID:22778705

Ki-67 and MCM-2 in dental follicle and odontogenic cysts: the effects of inflammation on proliferative markers.

PubMed

Güler, Nurhan; Comunoğlu, Nil; Cabbar, Fatih

2012-01-01

The aim of this study was to investigate whether there is any association between inflammation and the expression of markers of cell cycle entry (Ki-67 and MCM-2) in dental follicle (DF) of asymptomatic impacted teeth and odontogenic cysts. The study consisted of 70 DFs and 20 odontogenic cysts (radicular cyst (RC), dentigerous cyst (DC) and keratocytic odontogenic tumor (KCOT) located at posterior mandibular region. Histological findings of inflammation for all specimen and mucous cell prosoplasia, squamous metaplasia, glandular epithelium for all DFs were stained with hematoxyline and eosin, periodic acid schiff, alcian blue, and mucin. Epithelial cell proliferation was determined by using immunohistochemical labeling for Ki-67 and MCM-2. The histologic examinations showed 16% mucous cell prosoplasia, 54% squamous metaplasia, 20% glandular epithelium, 37% inflammation. Inflammation was detected in all RCs and %62 in DF, %43 in DC and KCOT. Positive correlation was found between the inflammation of DF and odontogenic cysts (P < 0.01). The mean Ki-67 and MCM-2 expressions were found 9, 64 ± 5, 99 and 6, 34 ± 3, 81 in DF, 11, 85 ± 9, 01 and 13, 6 ± 9, 94 in odontogenic cysts, respectively. While the mean Ki-67 expressions were statistically significant in DF and KCOT (P < 0.01), MCM-2 were significant in RC and KCOT (P < 0.01). MCM-2 expresion in RCs were statistically significant than KCOT (P < 0.01). The results of this study indicated that the higher MCM-2 expressions in RC than the KCOT might be related to the inflammation and this protein might be more sensitive to inflammation.

Repair of articular cartilage defects by tissue-engineered cartilage constructed with adipose-derived stem cells and acellular cartilaginous matrix in rabbits.

PubMed

Wang, Z J; An, R Z; Zhao, J Y; Zhang, Q; Yang, J; Wang, J B; Wen, G Y; Yuan, X H; Qi, X W; Li, S J; Ye, X C

2014-06-18

After injury, inflammation, or degeneration, articular cartilage has limited self-repair ability. We aimed to explore the feasibility of repair of articular cartilage defects with tissue-engineered cartilage constructed by acellular cartilage matrices (ACMs) seeded with adipose-derived stem cells (ADSCs). The ADSCs were isolated from 3-month-old New Zealand albino rabbit by using collagenase and cultured and amplified in vitro. Fresh cartilage isolated from adult New Zealand albino rabbit were freeze-dried for 12 h and treated with Triton X-100, DNase, and RNase to obtain ACMs. ADSCs were seeded in the acellular cartilaginous matrix at 2x10(7)/mL, and cultured in chondrogenic differentiation medium for 2 weeks to construct tissue-engineered cartilage. Twenty-four New Zealand white rabbits were randomly divided into A, B, and C groups. Engineered cartilage was transplanted into cartilage defect position of rabbits in group A, group B obtained ACMs, and group C did not receive any transplants. The rabbits were sacrificed in week 12. The restored tissue was evaluated using macroscopy, histology, immunohistochemistry, and transmission electron microscopy (TEM). In the tissue-engineered cartilage group (group A), articular cartilage defects of the rabbits were filled with chondrocyte-like tissue with smooth surface. Immunohistochemistry showed type II-collagen expression and Alcian blue staining was positive. TEM showed chondrocytes in the recesses, with plenty of secretary matrix particles. In the scaffold group (group B), the defect was filled with fibrous tissue. No repaired tissue was found in the blank group (group C). Tissue-engineered cartilage using ACM seeded with ADSCs can help repair articular cartilage defects in rabbits.

Curcumin alleviates lumbar radiculopathy by reducing neuroinflammation, oxidative stress and nociceptive factors.

PubMed

Xiao, L; Ding, M; Fernandez, A; Zhao, P; Jin, L; Li, X

2017-05-09

Current non-surgical treatments for lumbar radiculopathy [e.g. epidural steroids and Tumour necrosis factor-α (TNF-α) antagonists] are neither effective nor safe. As a non-toxic natural product, curcumin possesses an exceptional anti-inflammatory profile. We hypothesised that curcumin alleviates lumbar radiculopathy by attenuating neuroinflammation, oxidative stress and nociceptive factors. In a dorsal root ganglion (DRG) culture, curcumin effectively inhibited TNF-α-induced neuroinflammation, in a dose-dependent manner, as shown by mRNA and protein expression of IL-6 and COX-2. Such effects might be mediated via protein kinase B (AKT) and extracellular signal regulated kinase (ERK) pathways. Also, a similar effect in combating TNF-α-induced neuroinflammation was observed in isolated primary neurons. In addition, curcumin protected neurons from TNF-α-triggered excessive reactive oxygen species (ROS) production and cellular apoptosis and, accordingly, promoted mRNA expression of the anti-oxidative enzymes haem oxygenase-1, catalase and superoxide dismutase-2. Intriguingly, electronic von Frey test suggested that intraperitoneal injection of curcumin significantly abolished ipsilateral hyperalgesia secondary to disc herniation in mice, for up to 2 weeks post-surgery. Such in vivo pain alleviation could be attributed to the suppression, observed in DRG explant culture, of TNF-α-elicited neuropeptides, such as substance P and calcitonin gene-related peptide. Surprisingly, micro-computed tomography (μCT) data suggested that curcumin treatment could promote disc height recovery following disc herniation. Alcian blue/picrosirius red staining confirmed that systemic curcumin administration promoted regeneration of extracellular matrix proteins, visualised by presence of abundant newly-formed collagen and proteoglycan content in herniated disc. Our study provided pre-clinical evidence for expediting this natural, non-toxic pleiotropic agent to become a new and safe clinical treatment of radiculopathy.

Craniofacial abnormalities in homozygous Small eye (Sey/Sey) embryos and newborn mice.

PubMed

Kaufman, M H; Chang, H H; Shaw, J P

1995-06-01

The Small eye (Sey) gene in the mouse is lethal in the homozygous state. It is located on chromosome 2, is a mutation in the Pax-6 gene, and is genetically homologous with the human aniridia 2 (AN2) gene mutation. Numerous studies over the last few years, using genetic and molecular biological approaches, have investigated both the location of the gene as well as its possible mode of action. In the homozygous state, the primary defect appears to be limited to the failure of differentiation of the presumptive lens and nasal placodes. Such mice therefore display a characteristic phenotype; they possess neither eyes nor any nasal derivatives. Their heterozygous (Sey/+) and normal (+/+) littermates may be distinguished before birth only by a detailed examination of their eyes. Few detailed morphological/histological studies have been undertaken to date in the Sey/Sey embryos and newborn, and in the present study we describe a variety of craniofacial abnormalities that have not previously been reported. We observed, with one exception, delayed closure of the palate, and the presence in 80% of mice of an abnormal complement of upper incisor teeth, so that 35% possessed 1 supernumerary tooth while 45% possessed 2 supernumerary teeth. In these mice, a total of either 3 or 4, rather than the normal complement of 2, upper incisor teeth were present. Possibly the most unexpected finding, however, was the presence of a median cartilaginous rod-like structure which protruded between the 2 maxillae to give the Alizarin red S and Alcian blue-stained 'cleared' skulls of the newborn mice a characteristic 'unicorn-like' appearance. While this structure appeared to be a rostral extension of the chondrocranium, its exact derivation is unclear.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

PubMed

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-08-01

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. © The Author 2017. Published by Oxford University Press.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta

PubMed Central

Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A.; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

2017-01-01

Abstract Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. PMID:28475764

TRPA1 Channels Mediate Human Gingival Fibroblast Response to Phenytoin.

PubMed

López-González, M J; Luis, E; Fajardo, O; Meseguer, V; Gers-Barlag, K; Niñerola, S; Viana, F

2017-07-01

Drug-induced gingival enlargement (GE) is a frequent adverse effect observed in patients treated with anticonvulsant, immunosuppressant, and some antihypertensive medications-the antiepileptic phenytoin being the main drug associated with GE due to its high incidence (around 50%). The molecular mechanisms behind drug-induced gingival overgrowth are still unknown. By reverse transcription polymerase chain reaction, we demonstrate that the calcium-permeable ion channels TRPA1, TRPV1, and its capsaicin-insensitive isoform TRPV1b are expressed in human gingival fibroblasts (HGFs), the most abundant cellular type in periodontal tissue. Cultured HGFs responded with intracellular calcium elevations to phenytoin and to the canonical TRPA1 agonist allyl isothiocyanate. Application of phenytoin activated a nonselective cationic current in HGFs with a typical signature for TRPA1 channels. Moreover, this activation was blocked by HC030031, a specific TRPA1 blocker. Similarly, the use of shRNAs against hTRPA1 in HGFs reduced TRPA1 expression and activation by phenytoin. In addition, we show that phenytoin increased intracellular calcium levels in cells transfected with mouse or human TRPA1 channels. Responses to phenytoin were not observed in untransfected cells or cells expressing TRPM8 or TRPV1. The activation of HGFs by phenytoin was markedly reduced in the presence of antioxidant vitamins: ascorbic acid, folic acid, and α-tocopherol. By performing cell proliferation assays, we found that phenytoin did not augment the proliferation rate of HGFs. In contrast, alcian blue and picrosirius red staining of long-term HGFs cultures indicated that phenytoin induces extracellular matrix accumulation of collagen. Collectively, these findings support an important role of TRPA1 channels in phenytoin-induced GE, provide insight into the pathophysiologic mechanism, and offer novel therapeutic opportunities for its treatment.

Efficacy and Pharmacological Mechanism of Pronase-Enhanced Low-Dose Antibiotics for Helicobacter pylori Eradication

PubMed Central

Liu, Kai Y.; Du, Fang C.; Fu, Qiang; Zhang, Wei J.; Sun, Hong W.; Zhang, Yi; Gan, Le L.; Yue, Zhi Y.

2014-01-01

This study examined the efficacy and pharmacological mechanism of pronase-assisted low-dose antibiotics for eradication of Helicobacter pylori. Mongolian gerbils infected with H. pylori received 7-day treatment (omeprazole, different concentrations of pronase, amoxicillin, and clarithromycin), and the efficacy was assessed using the eradication rate and the colonization of H. pylori. In Mongolian gerbils orally administered pronase, the thickness of the gastric mucous layer (GML) was examined using immunohistochemical and alcian blue staining, and the concentrations of amoxicillin in gastric tissue and serum were detected using high-performance liquid chromatography (HPLC). The eradication rates were 80.0% (12/15) in the high-pronase quadruple group (HPQG) and 86.7% (13/15) in the high-antibiotic group (HAG) (P = 1.000). The antibiotic dose in the HPQG was only 1/20 that in the HAG. Thirty minutes after oral treatment with pronase, the sticky protein of the GML was hydrolyzed, and the GML became thinner. Higher amoxicillin concentrations in both the gastric tissue and serum were observed in the pronase group than in the Am10 group. The concentration of amoxicillin in the Am10-plus-Pr108 group in gastric tissue was 3.8 times higher than in the Am10 group in 5 min. Together, these data suggest that pronase significantly reduced the dose of antibiotics used in H. pylori eradication. The pharmacological mechanism is likely pronase removal of the mucus layer, promoting chemical factor (i.e., gastric acid and pepsinogen) distribution and increasing the antibiotic concentrations in the deep GML, which acted on H. pylori collectively. Thus, pronase may enhance the level of antibiotics for eradication of H. pylori in the clinic. PMID:24687504

No Beneficial Effect of General and Specific Anti-Inflammatory Therapies on Aortic Dilatation in Marfan Mice

PubMed Central

den Hartog, Alexander W.; Radonic, Teodora; de Vries, Carlie J. M.; Zwinderman, Aeilko H.; Groenink, Maarten; Mulder, Barbara J. M.; de Waard, Vivian

2014-01-01

Aims Patients with Marfan syndrome have an increased risk of life-threatening aortic complications, mostly preceded by aortic dilatation. In the FBN1 C1039G/+ Marfan mouse model, losartan decreases aortic root dilatation. We recently confirmed this beneficial effect of losartan in adult patients with Marfan syndrome. The straightforward translation of this mouse model to man is reassuring to test novel treatment strategies. A number of studies have shown signs of inflammation in aortic tissue of Marfan patients. This study examined the efficacy of anti-inflammatory therapies in attenuating aortic root dilation in Marfan syndrome and compared effects to the main preventative agent, losartan. Methods and Results To inhibit inflammation in FBN1 C1039G/+ Marfan mice, we treated the mice with losartan (angiotensin II receptor type 1 inhibitor), methylprednisolone (corticosteroid) or abatacept (T-cell-specific inhibitor). Treatment was initiated in adult Marfan mice with already existing aortic root dilatation, and applied for eight weeks. Methylprednisolone- or abatacept-treated mice did not reveal a reduction in aortic root dilatation. In this short time frame, losartan was the only treatment that significantly reduced aorta inflammation, transforming growth factor-beta (TGF-β) signaling and aortic root dilatation rate in these adult Marfan mice. Moreover, the methylprednisolone-treated mice had significantly more aortic alcian blue staining as a marker for aortic damage. Conclusion Anti-inflammatory agents do not reduce the aortic dilatation rate in Marfan mice, but possibly increase aortic damage. Currently, the most promising therapeutic drug in Marfan syndrome is losartan, by blocking the angiotensin II receptor type 1 and thereby inhibiting pSmad2 signaling. PMID:25238161

Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

PubMed

Yang, Hee Seok; La, Wan-Geun; Bhang, Suk Ho; Kim, Hak-Jun; Im, Gun-Il; Lee, Haeshin; Park, Jung-Ho; Kim, Byung-Soo

2011-07-01

Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.

CURCUMIN ALLEVIATES LUMBAR RADICULOPATHY BY REDUCING NEUROINFLAMMATION, OXIDATIVE STRESS AND NOCICEPTIVE FACTORS

PubMed Central

Xiao, L.; Ding, M.; Fernandez, A.; Zhao, P.; Jin, L.; Li, X.

2017-01-01

Current non-surgical treatments for lumbar radiculopathy [e.g. epidural steroids and Tumour necrosis factor-α (TNF-α) antagonists] are neither effective nor safe. As a non-toxic natural product, curcumin possesses an exceptional anti-inflammatory profile. We hypothesised that curcumin alleviates lumbar radiculopathy by attenuating neuroinflammation, oxidative stress and nociceptive factors. In a dorsal root ganglion (DRG) culture, curcumin effectively inhibited TNF-α-induced neuroinflammation, in a dose-dependent manner, as shown by mRNA and protein expression of IL-6 and COX-2. Such effects might be mediated via protein kinase B (AKT) and extracellular signal regulated kinase (ERK) pathways. Also, a similar effect in combating TNF-α-induced neuroinflammation was observed in isolated primary neurons. In addition, curcumin protected neurons from TNF-α-triggered excessive reactive oxygen species (ROS) production and cellular apoptosis and, accordingly, promoted mRNA expression of the anti-oxidative enzymes haem oxygenase-1, catalase and superoxide dismutase-2. Intriguingly, electronic von Frey test suggested that intraperitoneal injection of curcumin significantly abolished ipsilateral hyperalgesia secondary to disc herniation in mice, for up to 2 weeks post-surgery. Such in vivo pain alleviation could be attributed to the suppression, observed in DRG explant culture, of TNF-α-elicited neuropeptides, such as substance P and calcitonin gene-related peptide. Surprisingly, micro-computed tomography (µCT) data suggested that curcumin treatment could promote disc height recovery following disc herniation. Alcian blue/picrosirius red staining confirmed that systemic curcumin administration promoted regeneration of extracellular matrix proteins, visualised by presence of abundant newly-formed collagen and proteoglycan content in herniated disc. Our study provided pre-clinical evidence for expediting this natural, non-toxic pleiotropic agent to become a new and safe clinical treatment of radiculopathy. PMID:28485773

In-vivo characterization of a 3D hybrid scaffold based on PCL/decellularized aorta for tracheal tissue engineering.

PubMed

Ghorbani, Fariba; Moradi, Lida; Shadmehr, Mohammad Behgam; Bonakdar, Shahin; Droodinia, Atosa; Safshekan, Farzaneh

2017-12-01

As common treatments for long tracheal stenosis are associated with several limitations, tracheal tissue engineering is considered as an alternative treatment. This study aimed at preparing a hybrid scaffold, based on biologic and synthetic materials for tracheal tissue engineering. Three electrospun polycaprolactone (PCL) scaffolds, namely E1 (pure PCL), E2 (collagen-coated PCL) and E3 (PCL blended with collagen) were prepared. Allogeneic aorta was harvested and decellularized. A biodegradable PCL stent was fabricated and inserted into the aorta to prevent its collapse. Scaffold characterization results revealed that the 2-h swelling ratio of E2 was significantly higher than those of E1 and E3. In the first 3months, E2 and E3 exhibited almost equal degradabilities (significantly higher than that of E1). Moreover, tensile strengths of all samples were comparable with those of human trachea. Using rabbit's adipose-derived mesenchymal stem cells (AMSCs) and primary chondrocytes, E3 exhibited the highest levels of GAG release within 21days as well as collagen II and aggrecan expression. Fot the next step, AMSC-chondrocyte co-culture seeded scaffold was sutured to the acellular aorta, implanted into rabbits' muscle, and finally harvested after 4weeks of follow up. Harvested structures were totally viable due to the angiogenesis created by the muscle. H&E and alcian blue staining results revealed the presence of chondrocytes in the structure and GAG in the produced extracellular matrix. Since tracheal replacement using biologic and synthetic scaffolds usually results in tracheal collapse or granulation formation, a hybrid construct may provide the required rigidity and biocompatibility for the substitute. Copyright © 2017. Published by Elsevier B.V.

Rebamipide increases the amount of mucin-like substances on the conjunctiva and cornea in the N-acetylcysteine-treated in vivo model.

PubMed

Urashima, Hiroki; Okamoto, Takashi; Takeji, Yasuhiro; Shinohara, Hisashi; Fujisawa, Shigeki

2004-08-01

Rebamipide increases the amount of mucin-like substances in the stomach. We aimed to determine the effects of rebamipide on the amount of mucin-like substances in the conjunctiva and cornea of N-acetylcysteine-treated eyes. Furthermore, we attempted to evaluate the effects of rebamipide on the wound healing of N-acetylcysteine-treated eyes. The model was created by instilling 10% N-acetylcysteine solutions into rabbit eyes. Rebamipide was then applied on the day following the completion of N-acetylcysteine treatment. The amount of mucin-like substances on the conjunctiva and cornea was measured using the Alcian-blue binding method. The degree of damage was evaluated using scores based on the areas and densities of the cornea and conjunctival after staining using a rose Bengal solution under blind conditions. Rebamipide increased the level of mucin-like substances on the conjunctiva of N-acetylcysteine-treated eyes when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances covering the cornea. Moreover, 1% rebamipide improved the rose Bengal scores of the cornea and conjunctiva in N-acetylcysteine-treated eyes. Rebamipide increased mucin-like substances on the cornea and conjunctiva of N-acetylcysteine-treated eyes. In accordance with the mucin-increasing effects, rebamipide improved the rose Bengal scores for the cornea and conjunctiva of N-acetylcysteine-treated eyes. However, the relevance of these findings to dry eyes is unclear because it is not known whether the change in mucus expression in the N-acetylcysteine model is similar to what occurs in aqueous tear deficiency. Consequently, it may be worth trying on an animal model of keratoconjunctivitis sicca.

Temporospatial study of hexose transporters and mucin in the epithelial cells of chicken (Gallus gallus domesticus) small intestine.

PubMed

Hussar, P; Kaerner, M; Duritis, I; Plivca, A; Pendovski, L; Jaerveots, T; Popovska-Percinic, F

2017-12-01

The temporospatial patterns in the localization of hexose transporters as well as in the quantitative and qualitative differences of glycoprotein mucin produced by the goblet cells of broiler chicken (Gallus gallus domesticus) small intestine during their first postnatal month were studied. The integral membrane proteins glucose transporter-2 and -5 (GLUT-2 and GLUT-5) that facilitate the transport of hexoses across epithelial cell layers that separate distinct compartments in organism were detected in the chicken intestinal epithelial cells using immunohistochemical labeling with polyclonal primary antibodies Rabbit anti-GLUT-2 and Rabbit anti-GLUT-5 (IHC kit, Abcam, UK). The chemical composition of mucin (neutral, acid) was carried out by applying the histochemical reactions by Alcian-Blue and periodic acid-Schiff methods. The results revealed presence of the hexose transporters GLUT-2 and -5, immunolocalized in the enterocytes of broiler's small intestine and the temporospatial pattern of the density of goblet cells of intestinal mucosa as well as the chemical composition of mucin produced by the goblet cells in chicken immediately after hatching and in 30-days-old chicken's. Simultanously, when goblet cells remained unstained with both antibodies in intestinal epithelium in chicken of both ages or some moderate staining was noticed in 30-days-old chickens' ileal epithelium, the increase of neutral and acid mucin- containing cells per area unit in both segments of the small intestine was detected from the first day after hatching to 30 day of life and the densilty of goblet cells was found to be higher in ileal than in duodenal region. Copyright© by the Polish Academy of Sciences.

Gastroprotective effect of alpha-pinene and its correlation with antiulcerogenic activity of essential oils obtained from Hyptis species

PubMed Central

Pinheiro, Marcelo de Almeida; Magalhães, Rafael Matos; Torres, Danielle Mesquita; Cavalcante, Rodrigo Cardoso; Mota, Francisca Sheila Xavier; Oliveira Coelho, Emanuela Maria Araújo; Moreira, Henrique Pires; Lima, Glauber Cruz; Araújo, Pamella Cristina da Costa; Cardoso, José Henrique Leal; de Souza, Andrelina Noronha Coelho; Diniz, Lúcio Ricardo Leite

2015-01-01

Background: Alpha-pinene (α-pinene) is a monoterpene commonly found in essential oils with gastroprotective activity obtained from diverse medicinal plants, including Hyptis species. The genus Hyptis (lamiaceae) consists of almost 400 species widespread in tropical and temperate regions of America. In the north and northeastern Brazil, some Hyptis species are used in traditional medicine to treat gastrointestinal disturbances. Objective: The present study has investigated the gastoprotective effect of purified α-pinene in experimental gastric ulcer induced by ethanol and indomethacin in mice. Materials and Methods: Gastric ulcers were induced in male Swiss mice (20-30 g) by oral administration of absolute ethanol or indomethacin 45 min after oral pretreatment with vehicle, standard control drugs or α-pinene (10, 30, and 100 mg/kg). One hour after the ulcerative challenges, the stomach were removed, and gastric lesions areas measured. The effects of α-pinene on the gastric juice acidity were determined by pylorus ligation model. The gastrointestinal motility and mucus depletion were determined by measuring the gastric levels of phenol red and alcian blue, respectively. Hematoxylin and eosin stained sections of gastric mucosa of the experimental groups were used for histology analysis. Results: α-pinene pretreatment inhibited ethanol-induced gastric lesions, reduced volume and acidity of the gastric juice and increased gastric wall mucus (P < 0.05). Furthermore, we showed an interesting correlation between concentration of α-pinene and gastroprotective effect of Hyptis species (P Pearson = 0.98). Conclusion: Our data showed that the α-pinene exhibited significant antiulcerogenic activity and a great correlation between concentration of α-pinene and gastroprotective effect of Hyptis species was also observed. PMID:25709221

A priming effect of benthic gastropod mucus on sedimentary organic matter remineralization

NASA Astrophysics Data System (ADS)

Hannides, A. K.; Aller, R. C.

2016-02-01

Mucous gels are produced by benthic animals rapidly and in copious amounts, and have previously been shown to significantly affect diffusion rates of redox-sensitive ions and organic compounds in sediment pore waters. They are also a highly likely priming substrate whose addition in modest amounts affects sedimentary organic matter remineralization. We tested the priming effect of benthic infaunal mucus using secretions of the common gastropod Neverita duplicata as model substrate. Their composition is typical of marine molluscan mucus, consisting primarily of water (>96% by weight), which is in relative equilibrium with seawater. Salt-free dry weight constitutes 0.7% and 0.6% of total pedal and hypobranchial mucus, respectively. The C:N ratios of pedal and hypobranchial mucus indicate that the organic component consists of a mucopolysaccharide-glycoprotein complex that varies depending on its function, while low C:S ratios of the insoluble component and positive staining with Alcian Blue dye are indicative of S-ester and alkyl-SO42- groups bridging mucopolysaccharide and glycoprotein components. Anoxic incubations of pedal mucus of N. duplicata, sediment, and mucus-sediment mixture, resulted in the anaerobic generation of ΣCO2 and NH4+ at ratios lower than initial C:N ratios, indicating the preferential decomposition of N-rich moieties. Production rates of SCO2 and NH4+ in mucus-sediment incubations are higher, by 9±16% and 29±11%, respectively, than those predicted from linear addition of mucus-only and sediment-only rates. The statistically significant accelerated remineralization rate of N in the presence of modest mucus contribution (0.2% of total N), suggests that benthic mucus addition affects sedimentary organic matter remineralization processes through a "priming" effect.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

PubMed

Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.

PubMed

Kwizera, Richard; Akampurira, Andrew; Kandole, Tadeo K; Nielsen, Kirsten; Kambugu, Andrew; Meya, David B; Boulware, David R; Rhein, Joshua

2017-08-22

Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures. Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log 10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78-5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59-5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R 2  = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R 2  = 0.43, P = 0.4). Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.

Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage.

PubMed

Huo, Li-Jun; Ma, Xing-Hong; Yang, Zeng-Ming

2002-10-15

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.

Quirks of dye nomenclature. 1. Evans blue.

PubMed

Cooksey, C J

2014-02-01

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.

Methylene blue MMX® tablets for chromoendoscopy. Bioavailability, colon staining and safety in healthy volunteers undergoing a full colonoscopy.

PubMed

Di Stefano, A F D; Radicioni, M M; Vaccani, A; Fransioli, A; Longo, L; Moro, L; Repici, A

2018-06-01

Methylene blue-MMX® tablets are proposed as an aid for detection and visualisation of adenomas and carcinomas in patients undergoing colonoscopy, by improving their detection rate and highlighting the presence of the intestinal dysplastic lesions. Single total doses of 100 and 200 mg were administered to healthy volunteers undergoing a bowel cleansing preparation and a full colonoscopy to investigate the colonic staining. The pharmacokinetics of methylene blue and the safety after exposure to the tablets were also investigated. With 200 mg, the best staining, assessed as the sum of acceptable and good staining, was achieved in the ascending colon and rectosigmoid (75% subjects each), the transverse and the descending colon (approximately 63% each). Absence of staining or overstaining were reported for no colonic region of interest in any subject. Similar results were observed in the 100 mg dose group. Methylene blue blood concentrations reached a peak (C max ) in a median time (T max ) of 12 h with 100 mg and 16 h with 200 mg. AUC 0-t was 10.7 ± 6.7 μg/mLxh after 100 mg and 25.2 ± 7.4 μg/mLxh after 200 mg. Half-life ranged between 9 and 22 h after the lower dose and between 6 and 26 h after the higher dose. The cumulative excretion was about 28% after 100 mg and about 39% after 200 mg. The overall frequency of adverse events was 39%, but only one was related to the product: abnormal transaminases. The most frequent adverse event was a transient polyuria (17%). One serious adverse event (gastrointestinal haemorrhage) led the subject to study discontinuation and hospitalisation and another subject withdrew the study due to one adverse event (haematemesis). Either event was not related to methylene blue. Copyright © 2017. Published by Elsevier Inc.

Methodology of aniline blue staining of chromatin and the assessment of the associated nuclear and cytoplasmic attributes in human sperm.

PubMed

Sati, Leyla; Huszar, Gabor

2013-01-01

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this >80% overlap in staining patterns within the same spermatozoa.

Evaluation of Staining-Dependent Colour Changes in Resin Composites Using Principal Component Analysis

PubMed Central

Manojlovic, D.; Lenhardt, L.; Milićević, B.; Antonov, M.; Miletic, V.; Dramićanin, M. D.

2015-01-01

Colour changes in Gradia Direct™ composite after immersion in tea, coffee, red wine, Coca-Cola, Colgate mouthwash, and distilled water were evaluated using principal component analysis (PCA) and the CIELAB colour coordinates. The reflection spectra of the composites were used as input data for the PCA. The output data (scores and loadings) provided information about the magnitude and origin of the surface reflection changes after exposure to the staining solutions. The reflection spectra of the stained samples generally exhibited lower reflection in the blue spectral range, which was manifested in the lower content of the blue shade for the samples. Both analyses demonstrated the high staining abilities of tea, coffee, and red wine, which produced total colour changes of 4.31, 6.61, and 6.22, respectively, according to the CIELAB analysis. PCA revealed subtle changes in the reflection spectra of composites immersed in Coca-Cola, demonstrating Coca-Cola’s ability to stain the composite to a small degree. PMID:26450008

Evaluation of Staining-Dependent Colour Changes in Resin Composites Using Principal Component Analysis.

PubMed

Manojlovic, D; Lenhardt, L; Milićević, B; Antonov, M; Miletic, V; Dramićanin, M D

2015-10-09

Colour changes in Gradia Direct™ composite after immersion in tea, coffee, red wine, Coca-Cola, Colgate mouthwash, and distilled water were evaluated using principal component analysis (PCA) and the CIELAB colour coordinates. The reflection spectra of the composites were used as input data for the PCA. The output data (scores and loadings) provided information about the magnitude and origin of the surface reflection changes after exposure to the staining solutions. The reflection spectra of the stained samples generally exhibited lower reflection in the blue spectral range, which was manifested in the lower content of the blue shade for the samples. Both analyses demonstrated the high staining abilities of tea, coffee, and red wine, which produced total colour changes of 4.31, 6.61, and 6.22, respectively, according to the CIELAB analysis. PCA revealed subtle changes in the reflection spectra of composites immersed in Coca-Cola, demonstrating Coca-Cola's ability to stain the composite to a small degree.

Decorin content and near infrared spectroscopy analysis of dried collagenous biomaterial samples.

PubMed

Aldema-Ramos, Mila L; Castell, Joan Carles; Muir, Zerlina E; Adzet, Jose Maria; Sabe, Rosa; Schreyer, Suzanne

2012-12-14

The efficient removal of proteoglycans, such as decorin, from the hide when processing it to leather by traditional means is generally acceptable and beneficial for leather quality, especially for softness and flexibility. A patented waterless or acetone dehydration method that can generate a product similar to leather called Dried Collagenous Biomaterial (known as BCD) was developed but has no effect on decorin removal efficiency. The Alcian Blue colorimetric technique was used to assay the sulfated glycosaminoglycan (sGAG) portion of decorin. The corresponding residual decorin content was correlated to the mechanical properties of the BCD samples and was comparable to the control leather made traditionally. The waterless dehydration and instantaneous chrome tanning process is a good eco-friendly alternative to transforming hides to leather because no additional effects were observed after examination using NIR spectroscopy and additional chemometric analysis.

Copper Phthalocyanine-Functionalized Graphitic Carbon Nitride: A Hybrid Heterostructure toward Photoelectrochemical and Photocatalytic Degradation Applications.

PubMed

Liu, Zhong-Guo; Wan, Jia-Yun; Yang, Ze; Wang, Shi-Quan; Wang, Hang-Xing

2016-07-05

In this work, alcian blue 8GX (AB), a copper(II) phthalocyanine derivative, was employed to functionalize graphitic carbon nitride (g-C3 N4 ) for the preparation of a highly efficient photocatalyst. The approach relies on a facile AB-assisted ethanol/water mixed-solvent exfoliation of bulk g-C3 N4 . The as-prepared g-C3 N4 /AB hybrid possesses significantly enhanced solution dispersibility and photoelectrochemical performance resulting from the synergistic effect between g-C3 N4 and AB, which involves the optimization of intimate interfacial contact, extension of light absorption range, and enhancement of charge-transfer efficiency. This synergy contributes enormously to the photocatalytic degradation of rhodamine 6G (R6G) under light irradiation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amorphous material of the skin in amyotrophic lateral sclerosis: a morphologic and biochemical study

NASA Technical Reports Server (NTRS)

Ono, S.; Nagao, K.; Yamauchi, M.

1994-01-01

We performed morphologic studies on skin from seven patients with ALS and seven control subjects. By light microscopy, the wide spaces that separated collagen bundles reacted strongly with colloidal iron and alcian blue in ALS patients. Electron microscopy revealed markedly increased amorphous material that was positive for ruthenium red in the ground substance. These findings were not present in controls. Quantitative amino acid analysis showed that the amount of total amino acids (nmoles per mg dry weight) was significantly decreased (p < 0.01) in ALS patients compared with that of controls, and there was a significant negative correlation between skin amino acid content and duration of illness in ALS patients (r = -0.83, p < 0.001). These morphologic findings and biochemical data indicate that the amorphous material, which is markedly increased in ALS skin, includes glycosaminoglycans.

Lineage mapping and characterization of the native progenitor population in cellular allograft.

PubMed

Neman, Josh; Duenas, Vincent; Kowolik, Claudia; Hambrecht, Amanda; Chen, Mike; Jandial, Rahul

2013-02-01

The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells. To characterize the stem and progenitor population in cellular allograft, Osteocel Plus. To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts. Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and Osteocalcin; and (3) enzyme-linked immunosorbent assays (ELISA) for BMP-2, Osteocalcin, RANKL, Osteoprotegrin, and Osteocalcin. Clonal analysis of cells from cellular allograft was performed utilizing advance lentivirus lineage mapping techniques and massive parallel sequencing. Alizarin Red, Alcian Blue, and Oil red O staining assessed tripotential differentiation capacity. Serial trypsinization of allograft cellular bone matrix yielded approximately 1×105 cells per mL with viability greater than 90%. Cells expressed a panel of 84 MSC-associated genes in a pattern similar to but not identical to pure MSCs; specifically, 59 of 84 genes showed less than a 2.5-fold change in both cell types. Protein analysis showed that cellular allograft -derived cells maintained in nondifferentiation media expressed the early osteo-progenitor markers BMP-2, SMADs, and Runx2. Corresponding flow cytometry data for MSC markers revealed the presence of Stro-1 (49%), CD44 (99%), CD90 (42%), and CD146 (97%). Lineage mapping indicated that 62% of clones persisted and generated progeny through 10 passages, strongly suggesting the presence of bona fide stem cells. Passage 10 clones also exhibited tri-lineage differentiation capacity into osteogenic (Alizarin Red with H&E counterstain), chondrogenic (Alcian Blue), and adipogenic (Oil red O). Cells that did not proliferate through 10 passages presumably differentiated along an osteo-progenitor lineage. These data indicate that cellular allograft (Osteocel Plus) contains a heterogeneous population of cells with most cells demonstrating the capacity for extensive self-renewal and multipotential differentiation, which are hallmarks of stem cells. Whether stem cell-enriched allografts function comparably to autograft will require further studies, and their efficacy in facilitating arthrodesis will depend on randomized clinical studies. Copyright © 2013 Elsevier Inc. All rights reserved.

Using Perls Staining to Trace the Iron Uptake Pathway in Leaves of a Prunus Rootstock Treated with Iron Foliar Fertilizers

PubMed Central

Rios, Juan J.; Carrasco-Gil, Sandra; Abadía, Anunciación; Abadía, Javier

2016-01-01

The aim of this study was to trace the Fe uptake pathway in leaves of Prunus rootstock (GF 677; Prunus dulcis × Prunus persica) plants treated with foliar Fe compounds using the Perls blue method, which detects labile Fe pools. Young expanded leaves of Fe-deficient plants grown in nutrient solution were treated with Fe-compounds using a brush. Iron compounds used were the ferrous salt FeSO4, the ferric salts Fe2(SO4)3 and FeCl3, and the chelate Fe(III)-EDTA, all of them at concentrations of 9 mM Fe. Leaf Fe concentration increases were measured at 30, 60, 90 min, and 24 h, and 70 μm-thick leaf transversal sections were obtained with a vibrating microtome and stained with Perls blue. In vitro results show that the Perls blue method is a good tool to trace the Fe uptake pathway in leaves when using Fe salts, but is not sensitive enough when using synthetic Fe(III)-chelates such as Fe(III)-EDTA and Fe(III)-IDHA. Foliar Fe fertilization increased leaf Fe concentrations with all Fe compounds used, with inorganic Fe salts causing larger leaf Fe concentration increases than Fe(III)-EDTA. Results show that Perls blue stain appeared within 30 min in the stomatal areas, indicating that Fe applied as inorganic salts was taken up rapidly via stomata. In the case of using FeSO4 a progression of the stain was seen with time toward vascular areas in the leaf blade and the central vein, whereas in the case of Fe(III) salts the stain mainly remained in the stomatal areas. Perls stain was never observed in the mesophyll areas, possibly due to the low concentration of labile Fe pools. PMID:27446123

Efficacy of testicular sperm chromatin condensation assay using aniline blue-eosin staining in the IVF-ET cycle.

PubMed

Park, Yong-Seog; Kim, Myo Kyung; Lee, Sun-Hee; Cho, Jae Won; Song, In Ok; Seo, Ju Tae

2011-09-01

This study was performed to evaluate testicular sperm chromatin condensation using aniline blue-eosin (AB-E) staining and its effects on IVF-ET. Chromatin condensation was analyzed using AB-E staining in 27 cases of testicular sperm extraction. There were 19 cases of obstructive azoospermia (OA) and 8 cases of non-obstructive azoospermia (NOA) in IVF-ET. Mature sperm heads were stained red-pink whereas immature sperm heads were stained dark blue. The percentage of sperm chromatin condensation was calculated from the ratio of the number of red-pink sperm to the total number of sperm analyzed. The overall percentages of chromatin condensation in OA and NOA were 31.1±11.2% and 26.3±14.4%, respectively. The fertilization rate was significant higher in OA than NOA (p<0.05); however, the rates of good embryos and clinical pregnancy did not show statistical differences. In OA and NOA, statistical differences were not observed in the rate of chromatin condensation, fertilization, good embryos, and clinical pregnancy between the pregnant group and non-pregnant group. Chromatin condensation is less stable than OA and showed a low fertilization rate in NOA. While there were no significant differences in chromatin condensation results between NOA and OA, we propose that a pattern of decreased chromatin condensation in NOA is one of the factors of low fertilization results requiring further study.

A field method for making a quantitative estimate of altered tuff in sandstone

USGS Publications Warehouse

Cadigan, R.A.

1954-01-01

The use of benzidine to identify altered tuff in sandstone is practical for field or field laboratory studies associated with stratigraphic correlations, mineral deposit investigations, or paleogeographic interpretations. The method is based on the ability of saturated benzidine (C12H12N2) solution to produce a blue stain on montmorillonite-bearing tuff grains. The method is substantiated by the results of microscopic, X-ray spectrometer, and spectrographic tests which lead to the conclusion that: (1) the benzidine stain test differentiates grains of different composition, (2) the white or gray grains which are stained a uniform blue color are fragments of altered tuff, and (3) white or gray grains which stain in a few small spots are probably silicified tuff. The amount of sand grains taken from a hand specimen or an outcrop which will be held by a penny is spread out on a nonabsorbent white surface and soaked with benzidine for 5 minutes. The approximate number blue grains and the average grain size are used in a chart to determine a reference number which measures relative order of abundance. The chart, based on a volume relationship, corrects for the variation in the number of grains in the sample as the grain size varies. Practical use of the method depends on a knowledge of several precautionary measures as well as an understanding of the limitations of benzidine staining tests.

Ultrastructural Investigation of Pelvic Peritoneum in Patients With Chronic Pelvic Pain and Subtle Endometriosis in Association With Chromoendoscopy.

PubMed

Mehdizadehkashi, Abolfazl; Tahermanesh, Kobra; Fazel Anvari-Yazdi, Abbas; Chaichian, Shahla; Azarpira, Negar; Nobakht, Maliheh; Abed, Seyedeh Mehr; Hashemi, Neda

2017-01-01

To evaluate the pelvic peritoneum under chromoendoscopy by scanning electron microscopy (SEM) as well as light microscopy with hematoxylin and eosin staining and immunohistochemistry (IHC) assays in patients with chronic pelvic pain (CPP) associated with subtle endometriosis. Case series study (Canadian Task Force classification II). A referral academic community tertiary medical center. Three women aged 29 to 37 years were referred to the obstetrics and gynecology clinic of the tertiary university hospital with CPP. They were suspicious for endometriosis, were not responding to medical treatments, and had undergone previous pelvic laparoscopy to determine the stage of endometriosis and preparation of peritoneal samples under the guidance of staining with methylene blue in 0.25% dilution. Comparison of stained and unstained pelvic peritoneal samples after the instillation of 0.25% methylene blue into the pelvic cavity. In 3 patients, laparoscopic examination showed minimal endometriosis. A total of 18 samples (9 stained and 9 unstained) from the 3 patients were prepared for SEM. Ten of the samples (55.6%) showed microstructural peritoneal destruction (7 of 9 stained [77.7%] and 3 of 9 [33.4%] unstained). Eighteen samples (9 stained and 9 unstained) from the 3 patients were also prepared for IHC. Six of these samples (33.3%) were S-100-positive, including 4 of 9 (44.4%) stained samples and 2 of 9 (22.2%) unstained samples. In general, in the context of CPP and endometriosis, there is no established relationship between the severity of pain and stage of endometriosis. In the pathophysiology of CPP associated with endometriosis, ultrastructural changes can play a significant role. Under methylene blue staining, some destroyed areas were detected, but the stained areas do not necessarily correlate with increased microstructural peritoneal destruction. Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

PubMed

Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

2017-01-01

The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

PubMed

Greer, P W; Chandler, F W; Hicklin, M D

1980-06-01

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.

Safer staining method for acid fast bacilli.

PubMed Central

Ellis, R C; Zabrowarny, L A

1993-01-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol. Images PMID:7687254

Safer staining method for acid fast bacilli.

PubMed

Ellis, R C; Zabrowarny, L A

1993-06-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol.

Iron Stain on Wood

Treesearch

Mark Knaebe

2013-01-01

Iron stain, an unsightly blue–black or gray discoloration, can occur on nearly all woods. Oak, redwood, cypress, and cedar are particularly prone to iron stain because these woods contain large amounts of tannin-like extractives. The discoloration is caused by a chemical reaction between extractives in the wood and iron in steel products, such as nails, screws, and...

THE RELATIVE REACTION WITHIN LIVING MAMMALIAN TISSUES : I. GENERAL FEATURES OF VITAL STAINING WITH LITMUS.

PubMed

Rous, P

1925-02-28

The present paper is the first of a series of reports on the relative reaction of living tissues as determined by vital staining with indicators. It is possible to bring about a localized and a general coloration of living rats and mice with litmus. The animals remain in good health and the coloration of some of the tissues persists for months. Much of the dye is stored in cell granules, especially in those of the reticulo-endothelial elements, but a diffuse staining of certain tissues occurs, notably of bone, epidermis, cartilage, and connective tissue everywhere. In the intensity and localization of the bony coloration litmus has resemblances to madder. Diffuse staining with it renders blue most, if not all, of the tissues affected, while a granular staining causes others to become notably pink, owing to the fact that the indicator, though introduced into the organism in the blue form and circulating as such in the body fluids, is ordinarily red when stored in cells. The polymorphonuclear elements and macrophages of a peritoneal exudate, may become so laden with material colored red by litmus that the blue color of the fluid constituent is masked and the exudate appears a deep, turbid red. The phenomenon is but one manifestation of a notable acidity within cell granules throughout the organism. Like many another in the stained animals it would appear to be of physiological import. Some of the questions suggested by the work will be dealt with in the paper immediately following.

Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

PubMed

Lee, Hyo Seok; Cui, Lian; Li, Ying; Choi, Ji Suk; Choi, Joo-Hee; Li, Zhengri; Kim, Ga Eon; Choi, Won; Yoon, Kyung Chul

2016-01-01

To investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface. LEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2'7'-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed. TBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group. Overexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

Methylene blue dyeing of cellular nuclei during salpingoscopy, a new in-vivo method to evaluate vitality of tubal epithelium.

PubMed

Marconi, G; Quintana, R

1998-12-01

The Fallopian tube can be damaged by different noxious substances that may change cellular ultrastructure and function. Alteration of the cell membrane allows the passage of certain aniline dyes, which can stain the nucleus. A total of 310 Fallopian tubes from 163 patients who underwent a surgical or diagnostic laparoscopy during fertility studies was analysed by salpingoscopy. Cellular nuclei were stained by injection of 20 ml of a 10% solution of methylene blue in saline solution (NaCl 10%) through the cervical cannula prior to salpingoscopy. Evaluation of nuclear staining with methylene blue, adhesions, vascular alterations, and the flattening of folds in relation to pregnancy outcome was undertaken. Quantification of salpingoscopic findings was carried out according to a score. Flattening of folds and vascular alterations showed no difference in the pregnant and non-pregnant groups. On the other hand, adhesions and nuclear dyeing were significantly greater in the non-pregnant group (adhesions 13.6 versus 26.8%, P < 0.004, and nuclear dyeing: 25 versus 41.7%, P < 0.009, pregnant versus non-pregnant). Methylene blue dye is a new tool to evaluate in vivo cyto-histological tubal damage, and is a useful and simple method to provide a prognosis of salpingean function.

THE RELATIVE REACTION WITHIN LIVING MAMMALIAN TISSUES

PubMed Central

Rous, Peyton

1925-01-01

The present paper is the first of a series of reports on the relative reaction of living tissues as determined by vital staining with indicators. It is possible to bring about a localized and a general coloration of living rats and mice with litmus. The animals remain in good health and the coloration of some of the tissues persists for months. Much of the dye is stored in cell granules, especially in those of the reticulo-endothelial elements, but a diffuse staining of certain tissues occurs, notably of bone, epidermis, cartilage, and connective tissue everywhere. In the intensity and localization of the bony coloration litmus has resemblances to madder. Diffuse staining with it renders blue most, if not all, of the tissues affected, while a granular staining causes others to become notably pink, owing to the fact that the indicator, though introduced into the organism in the blue form and circulating as such in the body fluids, is ordinarily red when stored in cells. The polymorphonuclear elements and macrophages of a peritoneal exudate, may become so laden with material colored red by litmus that the blue color of the fluid constituent is masked and the exudate appears a deep, turbid red. The phenomenon is but one manifestation of a notable acidity within cell granules throughout the organism. Like many another in the stained animals it would appear to be of physiological import. Some of the questions suggested by the work will be dealt with in the paper immediately following. PMID:19868995

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice

PubMed Central

Doyle, Laura; Vogel, Sherilynn

2017-01-01

Abstract Background The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. Methods We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis-specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Results Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. Conclusion PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization. PMID:29062861

Primary adenocarcinomas of the human urinary bladder: histochemical, immunological and ultrastructural studies.

PubMed

Alroy, J; Roganovic, D; Banner, B F; Jacobs, J B; Merk, F B; Ucci, A A; Kwan, P W; Coon, J S; Miller, A W

1981-01-01

Neoplastic and non-neoplastic tissue specimens from ten patients with primary adenocarcinoma of the urinary bladder were examined. Most of these tumors were associated with either foci of transitional cell carcinoma and/or with glandular metaplasia of the bladder epithelium. The mucin produced by the neoplastic cells was PAS, alcian blue, mucicarmine, PB/KOH/PAS, and RPB/KOH/PAS-positive. ABH isoantigens of these tumors were not always deleted. Ultrastructurally, the neoplastic cells resembled goblet cells. Their plasma membrane had numerous microvilli with prominent glycocalyx. Proliferation and attenuation of tight junctions were noted. The gap junctions were few and small. Two types of desmosomes were found. The ultrastructural features of the neoplastic cells were attributed in part to the malignant transformation and in part to the direction of their differentiation. We have not observed any distinctive morphologic, histochemical, immunologic or ultrastructural features that might be diagnostic for these adenocarcinomas.

Decorin Content and Near Infrared Spectroscopy Analysis of Dried Collagenous Biomaterial Samples

PubMed Central

Aldema-Ramos, Mila L.; Castell, Joan Carles; Muir, Zerlina E.; Adzet, Jose Maria; Sabe, Rosa; Schreyer, Suzanne

2012-01-01

The efficient removal of proteoglycans, such as decorin, from the hide when processing it to leather by traditional means is generally acceptable and beneficial for leather quality, especially for softness and flexibility. A patented waterless or acetone dehydration method that can generate a product similar to leather called Dried Collagenous Biomaterial (known as BCD) was developed but has no effect on decorin removal efficiency. The Alcian Blue colorimetric technique was used to assay the sulfated glycosaminoglycan (sGAG) portion of decorin. The corresponding residual decorin content was correlated to the mechanical properties of the BCD samples and was comparable to the control leather made traditionally. The waterless dehydration and instantaneous chrome tanning process is a good eco-friendly alternative to transforming hides to leather because no additional effects were observed after examination using NIR spectroscopy and additional chemometric analysis. PMID:24970152

Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results

PubMed Central

Gebhard, Harry; Bowles, Robby; Dyke, Jonathan; Saleh, Tatianna; Doty, Stephen; Bonassar, Lawrence; Härtl, Roger

2010-01-01

Study type: Basic science Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3 b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)—Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics. Methods: Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: Bone Figure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc space Figure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a–c H&E (overall tissue staining for light micrsocopy) d–f Alcian blue (proteoglycans) g–i Picrosirius red (collagen I and II) Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body bone Figure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NP Figure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months. Conclusions: This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d–f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants. PMID:23637671

Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results.

PubMed

Gebhard, Harry; Bowles, Robby; Dyke, Jonathan; Saleh, Tatianna; Doty, Stephen; Bonassar, Lawrence; Härtl, Roger

2010-08-01

 Basic science Introduction:  Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)-Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective:  The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics.  Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: BoneFigure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc spaceFigure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a-c H&E (overall tissue staining for light micrsocopy) d-f Alcian blue (proteoglycans) g-i Picrosirius red (collagen I and II)Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body boneFigure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NPFigure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results:  The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months.  This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d-f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.

Altering the axial light gradient affects photomorphogenesis in emerging seedlings of Zea mays L

NASA Technical Reports Server (NTRS)

Parks, B. M.; Poff, K. L.

1986-01-01

The axial (longitudinal) red light gradient (632 nanometers) of 4 day old dark-grown maize seedlings is increased by staining the peripheral cells of the coleoptile. The magnitude of increase in the light gradient is dependent solely on the light-absorbing qualities of the stain used. Metanil yellow has no effect on the axial red-light gradient, while methylene blue causes a large increase in this light gradient. These stains did not affect growth in darkness or the sensitivity of mesocotyl elongation to red light. However, mesocotyl elongation was altered for the dark-grown seedlings stained with methylene blue when these seedlings were transplanted, covered with soil, and permitted to emerge under natural lighting conditions. These observations are consistent with the idea that there is a single perceptive site below the coleoptilar node, and suggest that this perceptive site gives the actinic light which has traveled downward through the length of the shoot from an entry point in the plant tip region.

Effect of autologous platelet-rich plasma on the chondrogenic differentiation of rabbit adipose-derived stem cells in vitro

PubMed Central

TANG, XIAO-BO; DONG, PEI-LONG; WANG, JIAN; ZHOU, HAI-YANG; ZHANG, HAI-XIANG; WANG, SHAN-ZHENG

2015-01-01

This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro. PMID:26622340

Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

PubMed

Serafini, R; Longobardi, V; Spadetta, M; Neri, D; Ariota, B; Gasparrini, B; Di Palo, R

2014-02-01

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates. © 2013 Blackwell Verlag GmbH.

Comparative study of efficacy, rapidity of detection, and cost-effectiveness of potassium hydroxide, calcofluor white, and Chicago sky blue stains in the diagnosis of dermatophytoses.

PubMed

Prakash, R; Prashanth, Hosakere Veerappa; Ragunatha, Shivanna; Kapoor, Meenakshi; Anitha, T K; Krishnamurthy, Veena

2016-04-01

The diagnosis of superficial mycosis such as dermatophytosis is often done clinically. However, in difficult cases, a rapid test with high sensitivity and specificity helps in the immediate confirmation and administration of treatment. The efficacy, rapidity of detection, and cost-effectiveness of KOH preparation, calcofluor white (CW) stain, and Chicago sky blue (CSB) stain in the identification of fungal elements were assessed in patients with dermatophytoses attending the dermatology clinic of a tertiary care hospital. All three tests were performed on each sample collected from 73 patients according to standard procedure. The slides were examined after 5 and 30 minutes in × 10 and × 40 magnifications. The sensitivity and specificity of CW and CSB at 5 and 30 minutes were calculated using KOH preparation as the standard test. CSB stain showed highest positivity (94.5%) within 5 minutes when compared to KOH (75.3%) and CW (83.5%). After 30 minutes, positivity increased in KOH (84.9%) and CW stains (89%), but it remained the same in CSB stain. Both CW and CSB stains when compared to 10% KOH are equally sensitive (100%), but CW was more specific (72.7%), particularly at 30 minutes. When cost of performing tests on 100 specimens is considered, KOH, CW, and CSB stains cost Rs 5, 100, and 15, respectively. CSB stain is a better stain for rapid diagnosis of dermatophytoses because of ease of performance, rapidity of detection, better appreciation of morphology of fungal elements, and cost effectiveness. © 2016 The International Society of Dermatology.

In vivo lymph node mapping by Cadmium Tellurium quantum dots in rats.

PubMed

Si, Chengshuai; Zhang, Yunpeng; Lv, Xianbo; Yang, Wuli; Ran, Zhipeng; Sun, Peng

2014-12-01

Intraoperative lymph node mapping (LNM) is highly significant for many surgeries in patients with cancer. Many types of tracers are currently used, but the ideal method has not yet been identified. We aimed to identify a stable lymphatic drainage pathway in an animal model and compared the effects of quantum dots (QD), a new fluorescent tracer, with those of methylene blue in intraoperative LNM. Indian ink (0.2 mL) was subcutaneously injected into the plantar metatarsal regions of six Sprague-Dawley rats. After 2 wk of incubation and subsequent dissection, the potentially stained LNs were examined pathologically to identify the lymphatic drainage pathway. After applying anesthesia, 0.1 mL methylene blue (2%) and QD (1 mg/mL) were injected into the plantar metatarsal regions of six rats for intraoperative LNM. The QD group was observed with a near-infrared imaging system, and the methylene blue group was directly observed. Drainages were recorded at 5, 10, 30, 60, and 120 min and at 1 d. Two three-level drainage pathways, that is, a peripheral drainage (popliteal LNs, inguinal LNs, and axillary LNs) and a central drainage (popliteal lymph node [LN], iliac LN, and renal LN) pathways were identified. Both methylene blue and QD stained the sentinel lymph node (SLNs) quickly, but methylene blue was difficult to identify in the deep tissues and the LNs beyond the SLN. Furthermore, the blue-stained LNs remain dyed for only 2 h. In contrast, the QDs exhibited high target-to-background ratios in both the SLNs and the following LNs. Additionally, the fluorescence lasted from 5 min-1 d after injection. An ideal lymphatic drainage model was found. QDs are excellent tracers for intraoperative LNM compared with methylene blue. Near infrared fluorescent imaging is a promising LNM method for clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

Surgical Marking Pen Dye Inhibits Saphenous Vein Cell Proliferation and Migration in Saphenous Vein Graft Tissue

PubMed Central

Kikuchi, Shinsuke; Kenagy, Richard D; Gao, Lu; Wight, Thomas N; Azuma, Nobuyoshi; Sobel, Michael; Clowes, Alexander W

2014-01-01

Objective Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Since little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This f cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either 1) % migration positive explants, which exclusively measures migration, or 2) the number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. Results There was significantly less cell migration from visibly blue, compared to unstained, outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein, because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in non-blue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9±8.0 ng dye/explant compared to 2.1±1.3 for non-blue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose-dependently inhibited migration (IC50=8.5 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/ml, while the EC50 for death was between 1 and 10 μg/ml. Conclusion Crystal violet inhibits venous cell migration and proliferation indicating that alternative methods should be considered for marking vein grafts. PMID:25935273

Determination of oestrous cycle of the rats by direct examination: how reliable?

PubMed

Yener, T; Turkkani Tunc, A; Aslan, H; Aytan, H; Cantug Caliskan, A

2007-02-01

For determination of the oestrous cycle in rats classical Papanicolaou technique has long been used successfully. Instead of using many stains in Papanicolaou, staining the vaginal secretions with only methylene blue has also been defined. Recently a new technique in which vaginal samples are directly examined under light microscope has been introduced. The aim of this study was to assess the reliability of this new technique by comparing it with the classical staining techniques. From 20 Wistar rats 60 vaginal samples were collected with a micropipette, three from each. Briefly, the vagina was flushed two to three times then the fluid was placed onto a glass slide. The fluid was equally distributed onto three glass slides. The glass slides were coded. Two samples were stained with Papanicolaou and methylene blue while the other one was examined directly. Determination of the phases of the oestrous cycle was made by the same histologist who was blinded to the groups and coding system. After determination of the oestrous phase in all samples, the results were compared and it was found that the results were matching. In conclusion, the same results can be obtained with the direct examination technique and this technique is reliable, so there is no need to use relatively time-consuming, less practical and more expensive techniques such as Papanicolaou or methylene blue.

The Ratio and Concentration of Two Monoterpenes Mediate Fecundity of the Pinewood Nematode and Growth of Its Associated Fungi

PubMed Central

Niu, Hongtao; Zhao, Lilin; Lu, Min; Zhang, Shuai; Sun, Jianghua

2012-01-01

The pinewood nematode (PWN) Bursaphelenchus xylophilus, vectored primarily by the sawyer beetle, Monochamus alternatus, is an important invasive pest and causal agent of pine wilt disease of Chinese Masson pine, Pinus massoniana. Previous work demonstrated that the ratios and concentrations of α-pinene∶β-pinene differed between healthy trees and those trees containing blue-stain fungus (and M. alternatus pupae). However, the potential influence of the altered monoterpene ratios and concentrations on PWN and associated fungi remained unknown. Our current results show that low concentrations of the monoterpenes within petri dishes reduced PWN propagation, whereas the highest concentration of the monoterpenes increased PWN propagation. The propagation rate of PWN treated with the monoterpene ratio representative of blue-stain infected pine (α-pinene∶β-pinene = 1∶0.8, 137.6 mg/ml) was significantly higher than that (α-pinene∶β-pinene = 1∶0.1, 137.6 mg/ml) representative of healthy pines or those damaged by M. alternatus feeding, but without blue stain. Furthermore, inhibition of mycelial growth of associated fungi increased with the concentration of the monoterpenes α-pinene and β-pinene. Additionally, higher levels of β-pinene (α-pinene∶β-pinene = 1∶0.8) resulted in greater inhibition of the growth of the associated fungi Sporothrix sp.2 and Ophiostoma ips strains, but had no significant effects on the growth of Sporothrix sp.1, which is the best food resource for PWN. These results suggest that host monoterpenes generally reduce the reproduction of PWN. However, PWN utilizes high monoterpene concentrations and native blue-stain fungus Sporothrix sp.1 to improve its own propagation and overcome host resistance, which may provide clues to understanding the ecological mechanisms of PWN's successful invasion. PMID:22363713

An in vitro analysis model for investigating the staining effect of various chlorhexidine-based mouthwashes.

PubMed

Kouadio, Alain-Ayepa; Struillou, Xavier; Bories, Céline; Bouler, Jean-Michel; Badran, Zahi; Soueidan, Assem

2017-03-01

There are different mouthwashes containing chlorhexidine in different concentrations, as well as various excipients. Chlorhexidine induce stains or discoloration in teeth and mucous membranes. The aim of this work was to design a model to reproduce in vitro staining associated with the use of different mouthwashes containing chlorhexidine. We used as substrates of natural teeth and elephant ivory slices. Different incubation baths were conducted over 21 days in culture dishes at 37°C. At the beginning of experiment before incubation (D0) and after 21 days (D21) of incubation with different mouthwashes, pictures of substrates were taken in a standardized manner and an image analysis software was used to analyse and quantify the staining under the various conditions by using the 3 main colours (Red, Green, Blue, RGB). The results of this work demonstrate a very good reproducibility of the protocol, and secondly, a different expression statistically significant of the primary blue colour. We suggest that for a given concentration of chlorhexidine, the staining effects may vary depending on the excipients used. This replicable model, easy to implement over a relatively short duration, can be used for evaluation of existing mouthwashes, and to test the excipients anti discoloration proposed by manufacturers. Key words: In vitro, chlorhexidine, mouthwashes, dental stain, tooth discoloration.

Effect of Photo-Fenton Bleaching on Tetracycline-stained Dentin in vitro.

PubMed

Bennett, Zackary Yale; Walsh, Laurence James

2015-02-01

Tetracycline-stained tooth structure is difficult to bleach using nightguard tray methods. The possible benefits of in-office light-accelerated bleaching systems based on the photo-Fenton reaction are of interest as possible adjunctive treatments. This study was a proof of concept for possible benefits of this approach, using dentine slabs from human tooth roots stained in a reproducible manner with the tetracycline antibiotic demeclocycline hydrochloride. Color changes overtime in tetra-cycline stained roots from single rooted teeth treated using gel (Zoom! WhiteSpeed(®)) alone, blue LED light alone, or gel plus light in combination were tracked using standardized digital photography. Controls received no treatment. Changes in color channel data were tracked overtime, for each treatment group (N = 20 per group). Dentin was lighter after bleaching, with significant improvements in the dentin color for the blue channel (yellow shade) followed by the green channel and luminosity. The greatest changes occurred with gel activated by light (p < 0.0001), which was superior to effects seen with gel alone. Use of the light alone did not significantly alter shade. This proof of concept study demonstrates that bleaching using the photo-Fenton chemistry is capable of lightening tetracycline-stained dentine. Further investigation of the use of this method for treating tetracycline-stained teeth in clinical settings appears warranted. Because tetracycline staining may respond to bleaching treatments based on the photo-Fenton reaction, systems, such as Zoom! WhiteSpeed, may have benefits as adjuncts to home bleaching for patients with tetracycline-staining.

The response of visible/near infrared absorbance to wood-staining fungi

Treesearch

Brian K. Via; Lori G. Eckhardt; Chi-Leung So; Todd F. Shupe; Leslie H. Groom; Michael Stine

2006-01-01

The influence of blue-stain fungi [Ophiostoma minus (Hedgcock) H. and P. Sydow and Leptographium serpens (Goid.) Siemaszko] on absorbance at the visible and near infrared wavelengths was investigated. Forty trees were sampled at breast height from longleaf pine (Pinus palustris Mill.). One half of each increment...

Methyl green and nitrotetrazolium blue chloride co-expression in colon tissue: A hyperspectral microscopic imaging analysis

NASA Astrophysics Data System (ADS)

Li, Qingli; Liu, Hongying; Wang, Yiting; Sun, Zhen; Guo, Fangmin; Zhu, Jianzhong

2014-12-01

Histological observation of dual-stained colon sections is usually performed by visual observation under a light microscope, or by viewing on a computer screen with the assistance of image processing software in both research and clinical settings. These traditional methods are usually not sufficient to reliably differentiate spatially overlapping chromogens generated by different dyes. Hyperspectral microscopic imaging technology offers a solution for these constraints as the hyperspectral microscopic images contain information that allows differentiation between spatially co-located chromogens with similar but different spectra. In this paper, a hyperspectral microscopic imaging (HMI) system is used to identify methyl green and nitrotetrazolium blue chloride in dual-stained colon sections. Hyperspectral microscopic images are captured and the normalized score algorithm is proposed to identify the stains and generate the co-expression results. Experimental results show that the proposed normalized score algorithm can generate more accurate co-localization results than the spectral angle mapper algorithm. The hyperspectral microscopic imaging technology can enhance the visualization of dual-stained colon sections, improve the contrast and legibility of each stain using their spectral signatures, which is helpful for pathologist performing histological analyses.

Epiretinal membrane negative staining and double peeling in a single block with Brilliant Blue G.

PubMed

Martins, David; Neves, Pedro

2018-01-01

To describe a surgical technique for combined peeling of epiretinal and internal limiting membranes. The authors present their procedure of choice for epiretinal membrane surgery: negative staining effect using Brilliant Blue G and single block removal of the epiretinal and internal limiting membranes in a single step. A total of 26 eyes were operated with the described technique. In all cases, the peeling was performed successfully and with no complications. Minimum postoperative follow-up was 12 months. There were no recurrences of epiretinal membranes. The ideal surgical approach for epiretinal membranes should attempt to reduce mechanical trauma, light exposure, and dye toxicity.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

A randomized, prospective cross-over trial comparing methylene blue-directed biopsy and conventional random biopsy for detecting intestinal metaplasia and dysplasia in Barrett's esophagus.

PubMed

Ragunath, K; Krasner, N; Raman, V S; Haqqani, M T; Cheung, W Y

2003-12-01

The value of methylene blue-directed biopsies (MBDB) in detecting specialized intestinal metaplasia and dysplasia in Barrett's esophagus remains unclear. The aim of this study was to compare the accuracy of MBDB with random biopsy in detecting intestinal metaplasia and dysplasia in patients with Barrett's esophagus. A prospective, randomized, cross-over trial was undertaken to compare MBDB with random biopsy in patients with Barrett's esophagus segments 3 cm or more in length without macroscopic evidence of dysplasia or cancer. Dysplasia was graded as: indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia, or carcinoma, and was reported in a blinded fashion. Fifty-seven patients were recruited, 44 of whom were male. A total of 1,269 biopsies were taken (MBDB-651, random biopsie-618). Analysis of the results by per-biopsy protocol showed that the MBDB technique diagnosed significantly more specialized intestinal metaplasia (75 %) compared to the random biopsy technique (68 %; P = 0.032). The sensitivity and specificity rates of MBDB for diagnosing specialized intestinal metaplasia were 91 % (95 % CI, 88 - 93 %) and 43 % (95 % CI, 36 - 51 %), respectively. The sensitivity and specificity rates of MBDB for diagnosing dysplasia or carcinoma were 49 % (95 % CI, 38 - 61 %) and 85 % (95 % CI, 82 - 88 %), respectively. There were no significant differences in the diagnosis of dysplasia and carcinoma - MBDB 12 %, random biopsy 10 %. The methylene blue staining pattern appeared to have an influence on the detection of specialized intestinal metaplasia and dysplasia/carcinoma. Dark blue staining was associated with increased detection of specialized intestinal metaplasia (P < 0.0001), and heterogeneous staining (P = 0.137) or no staining (P = 0.005) were associated with dysplasia and/or carcinoma detection. The MBDB technique prolonged the endoscopy examination by an average of 6 min. The diagnostic accuracy of the MBDB technique was superior to that of the random biopsy technique for identifying specialized intestinal metaplasia, but not dysplasia or carcinoma. The intensity of methylene blue staining has an influence on the detection of specialized intestinal metaplasia and dysplasia or carcinoma, which may help in targeting the biopsies. Although MBDB prolongs the endoscopy procedure slightly, it is a safe and well-tolerated procedure. Further clinical studies on the MBDB technique exclusively in endoscopically normal dysplastic Barrett's esophagus are needed.

Using methylene blue as a marker to find and remove tiny metallic foreign bodies embedded in the soft tissues of children: A randomised controlled trial.

PubMed

Su, Yuxi; Nan, Guoxin

2016-05-01

Embedment of metallic foreign bodies in the soft tissues is commonly encountered in the emergency room. Most foreign bodies are easily removed, but removal is difficult if the foreign body is very small or deeply embedded. To determine the usefulness of methylene blue staining in the surgical removal of tiny metallic foreign bodies embedded in the soft tissue. This prospective study involved 41 children treated between May 2007 and May 2012. The patients were randomly divided into a methylene blue group and a control group. In the control group, foreign bodies were located using a C-arm and removed via direct incision. In the methylene blue group, foreign bodies were located using a C-arm, marked with an injection of methylene blue and then removed surgically. The clinical outcomes, complications, operation time, surgical success rate, incision length, frequency of C-arm use, and length and depth of the foreign body were compared between the two groups. The surgical success rate was significantly higher in the methylene blue group. The average operation time was significantly shorter in the methylene blue group. The C-arm was used significantly less frequently in the methylene blue group than in the control group. The incision length was significantly shorter in the methylene blue group than in the control group. Methylene blue staining facilitated the location and removal of tiny metallic foreign bodies from the soft tissue, and significantly reduced operation time, incision length and radiation exposure compared to the conventional method. Copyright © 2016 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

[The prognostic value of gastric metaplasia in the duodenal mucosa in patients with Helicobacter pylori positive duodenal bulb ulcer].

PubMed

Marshalko, O V; Konorev, M R

2008-01-01

The predictive value of gastric metaplasia in the duodenal mucosa in patients Helicobacter pylori-positive patients with duodenal bulb ulcer (DBU) was investigated. One hundred and twenty four randomly selected patients with DBU were included in this prospective study. The detection of Helicobacter pylori (HP) in the stomach and duodenum was carried out with Giemsa (using standard visual analogue scale), rapid urease test (standard Jatrox-HP test, Rohm Pharma, Germany), and polymerase chain reaction (PCR) to detect the specific fragment of ureC HP gene (Helicopol II, Lytech, Russia). Regions of gastric metaplasia of the duodenum were confirmed by periodic acid-Schiff and alcian blue (Serva) staining (pH 1.0; 2.5) Duodenal ulcer (DU) complications were registered within 8 to 10 years. Estimation of the predictive factor (gastric metaplasia in the duodenum) was carried out in patients with non-complicated DU (Group 1; n = 73), and with such complications as bleeding, perforation, penetration, pyloroduodenal stenosis (Group 2; n = 51) which were revealed within the 8 to 10 years of observation. Gastric metaplasia in the duodenum was found in 64 or 87.7% of the 73 patients with non-complicated DU and in 5 or 9.8% of the 51 patients with complicated DU within 8 to 10 years of observation. The following facts about the predictive factor for the prognosis of DU complication were found: the sensitivity of 83.6%, the specificity of 92.8%, the predictive accuracy of 88.7%, the relative risk of the predicted outcome of 7.5, the relative risk of a different outcome of 0.11, the odds ration of 65.4. The study revealed a high and significant (p < 0.001) predictive value of gastric metaplasia in the duodenum as a marker of non-complicated clinical course of DU in HP-positive patients within an 8 to 10-year period.

Chronic Bronchitis and Current Smoking Are Associated with More Goblet Cells in Moderate to Severe COPD and Smokers without Airflow Obstruction

PubMed Central

Kim, Victor; Oros, Michelle; Durra, Heba; Kelsen, Steven; Aksoy, Mark; Cornwell, William D.; Rogers, Thomas J.; Criner, Gerard J.

2015-01-01

Background Goblet cell hyperplasia is a classic but variable pathologic finding in COPD. Current literature shows that smoking is a risk factor for chronic bronchitis but the relationship of these clinical features to the presence and magnitude of large airway goblet cell hyperplasia has not been well described. We hypothesized that current smokers and chronic bronchitics would have more goblet cells than nonsmokers or those without chronic bronchitis (CB), independent of airflow obstruction. Methods We recruited 15 subjects with moderate to severe COPD, 12 healthy smokers, and 11 healthy nonsmokers. Six endobronchial mucosal biopsies per subject were obtained by bronchoscopy and stained with periodic acid Schiff-Alcian Blue. Goblet cell density (GCD) was quantified as goblet cell number per millimeter of basement membrane. Mucin volume density (MVD) was quantified as volume of mucin per unit area of basement membrane. Results Healthy smokers had a greater GCD and MVD than nonsmokers and COPD subjects. COPD subjects had a greater GCD than nonsmokers. When current smokers (healthy smokers and COPD current smokers, n = 19) were compared with all nonsmokers (nonsmoking controls and COPD ex-smokers, n = 19), current smokers had a greater GCD and MVD. When those with CB (n = 12) were compared to those without CB (n = 26), the CB group had greater GCD. This finding was also seen in those with CB in the COPD group alone. In multivariate analysis, current smoking and CB were significant predictors of GCD using demographics, lung function, and smoking pack years as covariates. All other covariates were not significant predictors of GCD or MVD. Conclusions Current smoking is associated with a more goblet cell hyperplasia and number, and CB is associated with more goblet cells, independent of the presence of airflow obstruction. This provides clinical and pathologic correlation for smokers with and without COPD. PMID:25646735

Intestinal metaplasia with a high salt diet induces epithelial proliferation and alters cell composition in the gastric mucosa of mice.

PubMed

Xiao, Fang; Crissey, Mary Ann S; Lynch, John P; Kaestner, Klaus H; Silberg, Debra G; Suh, Eunran

2005-06-01

Intestinal metaplasia of the gastric mucosa is an important component in the pathway to adenocarcinoma. The mechanisms that induce the progression from intestinal metaplasia to cancer have not been elucidated. High dietary salt has been known as one of the risk factors for gastric cancer development in humans. Therefore, we investigated the role of high salt diet on gastric epithelial cell proliferation and differentiation, using our mouse model that ectopically expressed Cdx2 homeodomain transcription factor and induced an intestinal metaplastic phenotype in the gastric epithelia. Sixty Cdx2 transgenic and sixty age-matched wild-type littermates were studied. Fifty-percent Cdx2 transgenic and wild type mice were administered a high-salt diet and the other fifty-percent was fed a standard diet starting at 12 weeks after birth. At 10, 20 and 40 weeks after initiation of the diets, histopathological changes were determined by Hemotoxylin and Eosin, alcian blue, and periodic acid-Schiff (PAS) staining. Cell types and cell kinetics were assessed by immunohistochemistry. At 52 weeks, significant alterations in pathology were observed in the Cdx2 transgenic mice fed a high-salt diet, including elongation of gastric pits, reduction of the glandular zone in the gastric corpus, and deepening of glands in the antrum. In the Cdx2 transgenic mice fed a high salt diet, the parietal and chief cells were significantly decreased in the gastric corpus. A significant increase in cell proliferation and apoptosis in the corpus and antrum were observed in Cdx2 transgenic mice fed a high-salt diet as compared to wild-type littermates. Taken together, these data implicate that intestinal metaplasia in concert with a high-salt diet induces epithelial proliferation, apoptosis, and alters cellular types in the gastric mucosa of mice. Alteration in the composition of the gastric epithelium may play a role in influencing the microenvironment to engender susceptibility to carcinogens.

Leukemia/lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis.

PubMed

Liu, Chuan-ju; Prazak, Lisa; Fajardo, Marc; Yu, Shuang; Tyagi, Neetu; Di Cesare, Paul E

2004-11-05

Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia. We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P. S., Fang, C. H., Leslie, M. P., and Di Cesare, P. E. (2000) J. Orthop. Res. 18, 345-350; Issack, P. S., Liu, C. J., Prazak, L., and Di Cesare, P. E. (2004) J. Orthop. Res. 22, 751-758). In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein. LRF bound directly to the NRE both in vitro and in living cells. Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF. LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells. In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining. LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity. LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes.

PubMed

Yu, Seon-Mi; Kim, Song-Ja

2013-11-01

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes. © 2013 Published by Elsevier Inc.

Age-Related Adaptation of Bone-PDL-Tooth Complex: Rattus-Norvegicus as a Model System

PubMed Central

Leong, Narita L.; Hurng, Jonathan M.; Djomehri, Sabra I.; Gansky, Stuart A.; Ryder, Mark I.; Ho, Sunita P.

2012-01-01

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7±0.1 to 0.9±0.2 GPa) and cementum (0.6±0.1 to 0.8±0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated. PMID:22558292

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

PubMed Central

Henning, Tobias D; Sutton, Elizabeth J; Kim, Anne; Golovko, Daniel; Horvai, Andrew; Ackerman, Larry; Sennino, Barbara; McDonald, Donald; Lotz, Jeffrey; Daldrup-Link, Heike E

2010-01-01

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different Ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with Ferucarbotran using various protocols: Cells were labeled with 100μg Fe/ml for 4h and 18h and additional samples were cultured for 6 or 12 days after the 18-hour labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared to unlabeled controls by staining with alcian blue and hematoxylin & eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3T was investigated on day 1 and day 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T2-SE and T2*-GE-sequences. Iron uptake was significant for all labeling protocols (p< 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared to the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4h, inhibition of chondrogenesis was not significant. PMID:19670250

COMPARISON OF THE EFFECTS OF PAPAIN AND VITAMIN A ON CARTILAGE

PubMed Central

Thomas, Lewis; McCluskey, Robert T.; Potter, Jacobus L.; Weissmann, Gerald

1960-01-01

The administration of large amounts of vitamin A to rabbits has been shown to result in depletion of cartilage matrix. The normal basophilic, metachromatic, and Alcian blue staining properties of the matrix are lost, especially in articular and epiphyseal cartilage. The cartilage cells remain intact, but are reduced in size. These changes sometimes appeared as early as 48 hours after the initiation of daily injection of 1 million units of vitamin A, and were usually well established by 5 days. Some rabbits failed to show changes in cartilage, even after 5 daily injections. Increased amounts of material presumed to be chondroitin sulfate were present in the sera of vitamin A-treated rabbits, usually by 72 hours after the first injection. This was demonstrated by a turbidimetric procedure using hexamminecobaltic chloride. In rabbits given sulfur-35 (Na2S35O4) 5 days before the initiation of vitamin A treatment, it was shown that sulfur-35 was lost from articular and epiphyseal cartilage. This was associated with an increase in the non-dialyzable sulfur-35 in both serum and in the cobalt-precipitable material. These rabbits also excreted more sulfur-35 than rabbits not given vitamin A. There was a reduction in sulfur-35 activity in chondromucoprotein extracted from the ear cartilage of vitamin A-treated rabbits. The changes are interpreted as indicating that the administration of large amounts of vitamin A to rabbits results in removal of chondroitin sulfate from cartilage matrix. The administration of small amounts of crude papain causes histologic changes in cartilage that are remarkably similar to those seen in vitamin A-treated rabbits. The possibility is suggested that the changes in cartilage produced by administration of vitamin A to rabbits may be the result of activation of a proteolytic enzyme or enzymes, with properties similar to those of papain. PMID:13776507

Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

PubMed Central

Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

2013-01-01

Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to evaluate whether intra-articular injection of a mixture of bupivacaine and hyaluronan after arthroscopic surgery may protect against bupivacaine’s chondrotoxicity. PMID:22427617

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

PubMed Central

Huh, Jeong-Eun; Park, Yeon-Cheol; Seo, Byung-Kwan; Lee, Jae-Dong; Baek, Yong-Hyeon; Choi, Do-Young; Park, Dong-Suk

2013-01-01

We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105) cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone. PMID:23983790

Osteoarthritis and rheumatoid arthritis pannus have similar qualitative metabolic characteristics and pro-inflammatory cytokine response.

PubMed

Furuzawa-Carballeda, J; Macip-Rodríguez, P M; Cabral, A R

2008-01-01

Pannus in osteoarthritis (OA) has only recently been characterized. Little is known, however, regarding the behavior of OA pannus in vitro compared to rheumatoid arthritis (RA) pannus. The purpose of our study was to compare OA with RA pannus. Pannus and synovial tissue co-cultures from 5 patients with OA and 5 patients with RA obtained during arthroplasty were studied. Pannus was defined as the microscopic invasive granulation tissue covering the articular surface. Tissues were cultured for 7 days and stained with Alcian Blue technique. Interleukin-1beta (IL-1beta), IL-8, IL-10, IL-12, tumor necrosis factor-alpha (TNF-alpha), and interferon gamma (IFN-gamma) were also determined in supernatants by ELISA. Cartilage oligomeric matrix protein (COMP), type II collagen, TNF-alpha, IL-10 and Ki-67 expression were also detected by immunohistochemistry. All patients had vascular or fibrous pannus. Synovial proliferation, inflammatory infiltrates and a decrease of extracellular matrix proteins were observed in all tissue samples. Chondrocyte proliferation was lower in OA than RA cartilage. OA synovial tissue expressed lower levels of proteoglycans than RA synoyium. Type II collagen levels were lower in OA than in RA cartilage. Significantly higher levels of IL-1beta were found in the supernatants of RA pannus compared to OA pannus (p<0.05). High but similar levels of TNF-alpha, IL-8 and TIMP-1 were detected in OA and RA pannus supernatants. IL-10, IL-12 and IFN-gamma were undetectable. RA and OA pannus had similar pro-inflammatory and anti-inflammatory cytokine profile expression. OA cartilage, synovial tissue and pannus had lower production of proteoglycans, type II collagen and IL-1beta. It remains to be elucidated why OA pannus invades the cartilage surface but does not cause the marginal erosions typically seen in RA.

Mucosal barrier in ulcerative colitis and Crohn's disease.

PubMed

Dorofeyev, A E; Vasilenko, I V; Rassokhina, O A; Kondratiuk, R B

2013-01-01

Background. The mucus layer in the gastrointestinal tract plays important role in host innate defense, regulation of secretion, and absorption processes, maintaining colonization resistance, which composes the integrity of protective mucus barrier in the large intestine. Investigations of mucin expression in the colon mucosa can improve the understanding of protective function of mucosal barrier in ulcerative colitis (UC) and Crohn's disease (CD). Materials and Methods. 77 patients with UC and CD were examined. Histological analysis of colon mucosa was done by standard method (haematoxylin-eosin, alcian blue at pH 1.0 and 2.5 to determine sulfated and nonsulfated glycosaminoglycans and glycoproteins, and goblet cells). To characterize the mucus production the PAS-reaction was performed. Immunohistochemistry was performed using monoclonal mouse antibodies raised against MUC2, MUC3, MUC4, and TFF3 (USBiological, USA). Results. The moderate expression of MUC2 and MUC3 (50.0% and 32.1%, P = 0.03) and high expression of MUC4 and TFF3 in the colon mucosa were observed in all patients with CD. The intensive labeling of MUC4 and TFF3 occurred more often (42.9% and 57.1%, P = 0.03) in patients with CD. The level of expression of secretory MUC2 and transmembrane MUC3 and MUC4 in all patients with UC was low, up to its complete absence (59.2% and 53.1% cases, P = 0.05). TFF3 expression had high and medium staining intensity in patients with UC. Conclusions. Different types of mucins synthesis, secretion, and expression were found in patients with UC and CD. The expression of mucin MUC2, MUC3, MUC4, and TFF3 correlated with the activity of disease and the extent of the inflammatory process in the large intestine. The most pronounced alteration of mucins expression was observed in patients with severe UC and CD.

Apocynin protects against ethanol-induced gastric ulcer in rats by attenuating the upregulation of NADPH oxidases 1 and 4.

PubMed

El-Naga, Reem N

2015-12-05

Gastric ulcer is a common gastrointestinal disorder affecting many people all over the world. Absolute ethanol (5 ml/kg) was used to induce gastric ulceration in rats. Apocynin (50 mg/kg) was given orally one hour before the administration of absolute ethanol. Omeprazole (20 mg/kg) was used as a standard. Interestingly, apocynin pre-treatment provided 93.5% gastroprotection against ethanol-induced ulceration. Biochemically, gastric mucin content was significantly increased with apocynin pre-treatment. This finding was further supported by alcian blue staining of stomach sections obtained from the different treated groups. Also, gastric juice volume and acidity were significantly reduced. Apocynin significantly ameliorated ethanol-induced oxidative stress by replenishing reduced glutathione and superoxide dismutase levels as well as reducing elevated malondialdehyde levels in gastric tissues. Besides, ethanol-induced pro-inflammatory response was significantly decreased by apocynin pre-treatment via reducing elevated levels of pro-inflammatory markers; interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2 and inducible nitric oxide synthase. Additionally, caspase-3 tissue level was significantly reduced in apocynin pre-treated group. Interestingly, NADPH oxidase-1 (NOX-1) and NOX-4 up-regulation was shown to be partially involved in the pathogenesis of ethanol-induced gastric ulceration and was significantly reversed by apocynin pre-treatment. Gastroprotective properties of apocynin were confirmed by histopathological examination. It is worth mentioning that apocynin was superior in all aspects except gastric mucin content parameter where it was significantly increased by 13.5 folds in the omeprazole pre-treated group. This study was the first to show that apocynin is a promising gastroprotective agent against ethanol-induced gastric ulceration, partially via its anti-oxidant, anti-inflammatory, anti-apoptotic effects as well as down-regulating NOX-1 and NOX-4 expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development.

PubMed

Teraoka, Hiroki; Dong, Wu; Ogawa, Shuji; Tsukiyama, Shusaku; Okuhara, Yuji; Niiyama, Masayoshi; Ueno, Naoto; Peterson, Richard E; Hiraga, Takeo

2002-02-01

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on regional red blood cell (RBC) perfusion rate, as an index of blood flow, and lower jaw development were investigated quantitatively in zebrafish embryos (Danio rerio) during early development. As revealed by observation of live embryos and alcian-blue staining, TCDD retarded lower jaw development in a concentration-dependent manner with only a minor inhibitory effect on total body length. Both inhibitory effects were significant as early as 60 h postfertilization (hpf), at which time the area of goosecoid (gsc) mRNA expression was clearly reduced in the lower jaw. To examine effects of TCDD on RBC perfusion rate, time-lapse recording was performed using a digital video camera attached to a light microscope. TCDD did not show marked effects on RBC perfusion rate until 72 hpf, when vessel-specific effects emerged. TCDD severely inhibited RBC perfusion rate in intersegmental arteries of the trunk, but only modestly and slightly inhibited RBC perfusion rate in certain vessels of the head such as the central arteries and optic vein. Conversely, at both 72 and 84 hpf, TCDD significantly increased RBC perfusion rate in the hypobranchial artery branching to the lower jaw primordia, and then reduced it at 96 hpf. RBC perfusion rate in all vessels examined in TCDD-exposed embryos was inhibited at 96 hpf. The zebrafish aryl hydrocarbon receptor 2 (zfAhR2) mRNA was strongly expressed in the lower jaw primordia at 48 hpf, and expression of this transcript was augmented by TCDD treatment. Thus, TCDD exposure of the zebrafish embryo has a disruptive effect on local circulation and lower jaw cartilage growth. Initially, TCDD may act directly on the lower jaw primordia to impair lower jaw development. Reductions in hypobranchial RBC perfusion rate occurred well after the initial retardation in lower jaw development had become apparent, and may contribute further to the effect.

Individual marking of soft-bodied subtidal invertebrates in situ – A novel staining technique applied to the giant plumose anemone Metridium farcimen (Tilesius, 1809)

PubMed Central

Sebens, Kenneth P.

2017-01-01

The ability to recognize individuals and track growth over time is crucial to population dynamics research as well as studies of animal behavior. Invertebrates are particularly difficult to track as they often molt, have regenerative capabilities, or lack hard parts to attach markers. We tested, in laboratory and field studies, a new way of marking sea anemones (order Actiniaria) by injection of three vital stains (i.e., neutral red, methylene blue, and fluorescein). Neutral red and methylene blue did not affect growth or survival, but fluorescein was lethal at high concentrations. Marked individuals could be identified up to seven months after injection with neutral red, six weeks with methylene blue, and three days with low concentrations of fluorescein. Neutral red could be used for long-term monitoring of growth and survival in the field, and in combination with methylene blue could be used to mark individuals in distinguishable patterns for short-term studies such as examining predator-prey interactions, movement of individuals, and recruitment survival. PMID:29161292

Prognostic value of the frequency of vascular invasion in stage I non-small cell lung cancer.

PubMed

Okada, Satoshi; Mizuguchi, Shinjiro; Izumi, Nobuhiro; Komatsu, Hiroaki; Toda, Michihito; Hara, Kantaro; Okuno, Takahiro; Shibata, Toshihiko; Wanibuchi, Hideki; Nishiyama, Noritoshi

2017-01-01

There is no standard pathological method for determining vessel invasion in lung cancer. Herein, we examine whether vessel invasion can be accurately assessed using hematoxylin-eosin staining alone, and investigate the prognostic impact of the presence and frequency of vessel invasion in lung cancer. Vessel invasion was assessed by hematoxylin-eosin, Victoria blue, and D2-40 in 251 completely resected stage I non-small cell lung cancer patients. Vessel invasion was classified into 3 grades according to the number of invaded vessels. Using hematoxylin-eosin and Victoria blue, vascular invasion was detected in 27 (10.8 %) and 75 (29.9 %) of patients, respectively. Lymphatic permeation was detected in 126 (50.2 %) and 70 (27.9 %) of patients using hematoxylin-eosin and D2-40 staining. Hematoxylin-eosin staining did not accurately detect a high frequency of vessel invasion; only 5 and 21.7 % of high-frequency vascular invasion and lymphatic permeation cases diagnosed with Victoria blue and D2-40 were detected. Multivariate analysis based on elastic stain and immunostaining indicated that plural invasion, a high frequency of vascular invasion (hazard ratio 4.00), and a high frequency of lymphatic permeation (hazard ratio 2.30) were independent predictors of cancer recurrence within 3 years. Likewise, an age ≥70 years, male, and a high frequency of vascular invasion (hazard ratio 3.41) were independent predictors of overall survival. Vascular invasion should be confirmed by elastic stains, and the frequency, not but the presence, of vascular invasion is a powerful independent prognostic factor in completely resected stage I non-small cell lung cancer patients.

White Light–Emitting Diodes (LEDs) at Domestic Lighting Levels and Retinal Injury in a Rat Model

PubMed Central

Shang, Yu-Man; Wang, Gen-Shuh; Sliney, David; Lee, Li-Ling

2013-01-01

Background: Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000–10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model. Objective: We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements. Methods: We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level. Results: H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury. Conclusion: Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich “white” LEDs for general lighting. Citation: Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light–emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269–276; http://dx.doi.org/10.1289/ehp.1307294 PMID:24362357

'Blue bubble' technique: an ab interno approach for Descemet separation in deep anterior lamellar keratoplasty using trypan blue stained viscoelastic device.

PubMed

Livny, Eitan; Bahar, Irit; Hammel, Naama; Nahum, Yoav

2018-04-01

In this study, we examined a novel variant of 'big-bubble' deep anterior lamellar keratoplasty using trypan-blue-stained viscoelastic device for the creation of a pre-descemetic bubble. Ten corneoscleral rims were mounted on an artificial anterior chamber (AC). The AC was filled with air through a limbal paracentesis. A Melles' triangulated spatula was inserted through the paracentesis, with its tip penetrating the AC, was then slightly retracted and pushed into the deep stroma above the roof of the paracentesis. A mixture of trypan blue and viscoelastic device (Healon, Abbott Medical Optics, Abbott Park, Illinois) was injected into this intra-stromal pocket using a 27-G cannula to create a pre-descemetic separation bubble. Bubble type and visualization of dyed viscoelastic device were noted. The method was later employed in three cases. In all 10 corneoscleral rims, the technique successfully created a visible pre-descemetic (type 1) bubble that could be expanded up to the predicted diameter of trephination. Subsequent trephination and the removal of corneal stroma were uneventful. In two out of four clinical cases, a type 1 bubble was created, while in two others, visco-dissection failed and dyed viscoelastic was seen in the AC. The presented technique holds promise of being a relatively easy to perform, predictable and well-controlled alternative for achieving a type 1 bubble during deep anterior lamellar keratoplasty surgery. The trypan-blue-stained viscoelastic device facilitates proper visualization and control of the separation bubble and assists in identifying the penetrance to the separation bubble prior to removal of the stromal cap. © 2017 Royal Australian and New Zealand College of Ophthalmologists.

Automated selection of the most epithelium-rich areas in gynecologic tumor sections.

PubMed

Schipper, N W; Baak, J P; Smeulders, A W

1991-12-01

The paper describes an image analysis technique for automated selection of the epithelium-rich areas in standard paraffin tissue sections of ovarian and endometrial premalignancies and malignancies. Two staining procedures were evaluated, Feulgen (pararosanilin) and CAM 5.2, demonstrating the presence of cytokeratin 8 and 18; both were counterstained with naphthol yellow. The technique is based on the corresponding image processing method of automated estimation of the percentage of epithelium in interactively selected microscope fields. With the technique, one image is recorded with a filter to demonstrate where epithelium and stroma lie. This filter is chosen according to the type of staining: it is yellow (lambda = 552 nm) for Feulgen and blue (lambda = 470 nm) for anticytokeratin CAM 5.2. When stroma cannot be distinguished from lumina with the green filter or from epithelium with the blue filter, a second image is recorded from the same microscope field, with a blue filter (lambda = 420 nm) for Feulgen and a yellow filter (lambda = 576 nm) for anticytokeratin CAM 5.2. Discrimination between epithelium and stroma is based on the image contrast range and the packing of nuclei in the yellow image and on the automated classification of the gray value histogram peaks in the blue image. For Feulgen stain the method was evaluated on 30 ovarian tumors of the common epithelial types (8 borderline tumors and 22 carcinomas with various degrees of differentiation) and 30 endometrial carcinomas of different grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Delineation of the vitreous and posterior hyaloid using bromophenol blue.

PubMed

Haritoglou, Christos; Strauss, Rupert; Priglinger, Siegfried G; Kreutzer, Thomas; Kampik, Anselm

2008-02-01

To describe visualization of the vitreous and the posterior hyaloid membrane using bromophenol blue during vitrectomy for macular hole and retinal detachment. Six patients with macular holes and four with retinal detachments were included in the study. Before and after surgery, complete clinical examination, including funduscopy and measurements of best-corrected visual acuity and intraocular pressure, was performed. Additional functional tests, such as fluorescein angiography, optical coherence tomography (Stratus OCT; Carl Zeiss Meditec, Jena, Germany, Germany), Goldmann perimetry, and multifocal electroretinography as well as photography of the posterior pole, were performed for macular hole patients. Bromophenol blue was used in concentrations of 0.2%. During macular hole surgery, the dye was injected into the air-filled globe, while during surgery for retinal detachment, the globe was partially filled with perfluorocarbon before dye injection after induction of a posterior vitreous detachment to stain the vitreous peripherally. Bromophenol blue provided sufficient staining of the attached posterior hyaloid membrane and vitreous remnants in the periphery. This was especially helpful for patients in whom a posterior vitreous detachment could not be induced mechanically by suction using the vitrectomy probe alone, as seen in three of six interventions for a macular hole in this series. In addition, staining of the vitreous or vitreous remnants in the periphery and at the vitreous base was seen in all patients and helped to completely remove the vitreous in a controlled fashion. After macular hole surgery, increase of visual acuity from 20/100 (mean) to 20/40 was seen during follow-up up to 6 months. In one case, the hole persisted and required a second operation. Finally, closure of the hole was achieved in all patients. After retinal detachment surgery, reattachment was achieved in all cases. No dye-related adverse events were seen during follow-up as shown by the functional tests (visual acuity measurement, electroretinography, and perimetry) applied. Delineation of the vitreous and the posterior hyaloid using bromophenol blue staining greatly facilitates vitreoretinal procedures. Bromophenol blue appeared to be a very helpful and safe tool to visualize the posterior hyaloid membrane in macular hole surgery and assured its complete separation from the retinal surface. The dye also helped to remove vitreous at the vitreous base during retinal detachment surgery. Therefore, bromophenol blue appears as a very good alternative to triamcinolone, which has been used for this purpose, because the dye has no pharmacological properties and no side effects are likely to occur such as cataract formation and increase in intraocular pressure. Further studies including larger numbers of patients are mandatory.

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

PubMed

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Differentiation of histoplasma and cryptococcus in cytology smears: a diagnostic dilemma in severely necrotic cases.

PubMed

Ranjan, R; Jain, D; Singh, L; Iyer, V K; Sharma, M C; Mathur, S R

2015-08-01

The correct identification of fungal organisms is important for the appropriate clinical management of patients. It becomes difficult in necrotic smears when the tissue response is not clearly discernible. It is difficult to distinguish between histoplasma and cryptococcus in severely necrotic cases, where both appear as variably sized clear refractile haloes. Four cases of adrenal necrotic histoplasma infection were studied and the morphology was compared with that of non-necrotic histoplasmosis and cases of cryptococcal infection. Eleven cases were analysed in fine needle aspiration cytology (FNAC) smears. Ziehl-Neelsen (ZN) stain was performed to exclude tuberculosis in necrotic smears. A clinical and serology correlation was performed where available. Necrotic cases of histoplasma infection revealed negative refractile clear haloes similar to those of cryptococcus. Histoplasma showed methylene blue-stained organisms in ZN stains, whereas the cryptococcus cases were negative. Similar methylene blue-stained organisms were seen in non-necrotic histoplasma infection. As a result of morphological overlap between cryptococcus and histoplasma, the distinction between the two fungi can be difficult in many cases. ZN staining appears to have a role in the differentiation of these fungi in severely necrotic cases. This observation needs to be validated on a larger number of cases with complete correlation with clinical, serology and treatment records. © 2014 John Wiley & Sons Ltd.

Detection of Pneumocystis jirovecii by nested PCR in HIV-negative patients with pulmonary disease.

PubMed

Santos, Cristina Rodrigues; de Assis, Ângela M; Luz, Edson A; Lyra, Luzia; Toro, Ivan F; Seabra, José Claudio C; Daldin, Dira H; Marcalto, Tathiane U; Galasso, Marcos T; Macedo, Ronaldo F; Schreiber, Angélica Z; Aoki, Francisco H

Nested PCR can be used to determine the status of Pneumocystis jirovecii infection in other lung diseases. This study sought to detect a target DNA fragment (mitochondrial large subunit rRNA or mtL SUrRNA) of P. jirovecii in patients with lung disease who underwent bronchoscopy with collection of bronchoalveolar lavage (BAL). The results from toluidine blue staining were compared with those obtained using molecular methods that included an "in house" DNA extraction procedure, PCR and nested PCR. Fifty-five BAL samples from patients with atypical chest X-rays were screened for P. jirovecii. None of the samples was positive for P. jirovecii using toluidine blue staining. In contrast, P. jirovecii DNA was detected by nested PCR in BAL samples from 36 of 55 patients (65.5%). The lung diseases in the patients included cancer, pneumonia, tuberculosis, and chronic obstructive pulmonary disease (COPD). Other chronic problems in the patients included hypertension, diabetes, smoking, and alcoholism. Nested PCR showed high sensitivity for detecting P. jirovecii, especially when compared with toluidine blue staining. Using this method, P. jirovecii infection was detected in HIV-negative patients with lung disease. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

DEVELOPMENT OF A MODEL TO INVESTIGATE RED BLOOD CELL SURFACE CHARACTERISTICS AFTER CRYOPRESERVATION.

PubMed

Gordiyenko, O I; Anikieieva, M O; Rozanova, S L; Kovalenko, S Ye; Kovalenkol, I F; Gordiyenko, E O

2015-01-01

Maintaining cell surface properties after freezing and thawing, characterized in particular by the surface potential and associated with it cell ability to intercellular adhesion, could be used as a characteristic of successful cryopreservation. This study was conducted to research applying different erythrocytes freezing modes and analyses the regimes cryopreservation effect on the cell surface charge and adhesion to microorganisms. Human erythrocytes frozen by three modes. In order to determine adhesion index was used dried bacterial cells of S. thermophilus. The surface charge of erythrocytes was evaluated using Alcian blue cationic dye. The results showed the significant decrease in the lactobacillus adhesion to erythrocytes frozen glycerol and 1,2-propanediol. After erythrocytes were freezen with glycerol and 1,2-propanediol, the cationic dye binding to erythrocytes significantly reduced. AB binding to erythrocytes frozen with PEG-1500 does not differ from control data. Erythrocytes frozen with PEG-1500 mantained surface properties after thawing better, compared to erythrocytes cryopreserved by other methods.

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

PubMed

Cong, Wei-Tao; Wang, Xu; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

2012-01-01

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Multispectral image enhancement for H&E stained pathological tissue specimens

NASA Astrophysics Data System (ADS)

Bautista, Pinky A.; Abe, Tokiya; Yamaguchi, Masahiro; Ohyama, Nagaaki; Yagi, Yukako

2008-03-01

The presence of a liver disease such as cirrhosis can be determined by examining the proliferation of collagen fiber from a tissue slide stained with special stain such as the Masson's trichrome(MT) stain. Collagen fiber and smooth muscle, which are both stained the same in an H&E stained slide, are stained blue and pink respectively in an MT-stained slide. In this paper we show that with multispectral imaging the difference between collagen fiber and smooth muscle can be visualized even from an H&E stained image. In the method M KL bases are derived using the spectral data of those H&E stained tissue components which can be easily differentiated from each other, i.e. nucleus, cytoplasm, red blood cells, etc. and based on the spectral residual error of fiber weighting factors are determined to enhance spectral features at certain wavelengths. Results of our experiment demonstrate the capability of multispectral imaging and its advantage compared to the conventional RGB imaging systems to delineate tissue structures with subtle colorimetric difference.

High-content analysis of single cells directly assembled on CMOS sensor based on color imaging.

PubMed

Tanaka, Tsuyoshi; Saeki, Tatsuya; Sunaga, Yoshihiko; Matsunaga, Tadashi

2010-12-15

A complementary metal oxide semiconductor (CMOS) image sensor was applied to high-content analysis of single cells which were assembled closely or directly onto the CMOS sensor surface. The direct assembling of cell groups on CMOS sensor surface allows large-field (6.66 mm×5.32 mm in entire active area of CMOS sensor) imaging within a second. Trypan blue-stained and non-stained cells in the same field area on the CMOS sensor were successfully distinguished as white- and blue-colored images under white LED light irradiation. Furthermore, the chemiluminescent signals of each cell were successfully visualized as blue-colored images on CMOS sensor only when HeLa cells were placed directly on the micro-lens array of the CMOS sensor. Our proposed approach will be a promising technique for real-time and high-content analysis of single cells in a large-field area based on color imaging. Copyright © 2010 Elsevier B.V. All rights reserved.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xin; Chen, Jian-long; Ma, Zheng-lai

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10{sup −8}–10{sup −6} μmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly,more » histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development. - Highlights: • Chick embryos occurred shortening of the long bone following Dex exposure. • Dex suppressed chondrocytes proliferation and promoted apoptosis. • Dex exposure decreased ALP production and up-regulated Runx-2 and Mmp-13. • Dex exhibited biphasic effects on chondrogenic proliferation and nodule formation. • The hypertrophy and ossification were accelerated by Dex both in vivo and in vitro.« less

Methylene blue-related corneal edema and iris discoloration.

PubMed

Timucin, Ozgur Bulent; Karadag, Mehmet Fatih; Aslanci, Mehmet Emin; Baykara, Mehmet

2016-04-01

We report the case of a 70-year-old female patient who developed corneal edema and iris discoloration following the inadvertent use of 1% methylene blue instead of 0.025% trypan blue to stain the anterior capsule during cataract phacoemulsification surgery. Copious irrigation was performed upon realization of incorrect dye use. Corneal edema and iris discoloration developed during the early postoperative period and persisted at 24-months follow-up. However, keratoplasty was not required. The intracameral use of 1% methylene blue has a cytotoxic effect on the corneal endothelium and iris epithelium. Copious irrigation for at least 30 min using an anterior chamber maintainer may improve outcomes.

Influence of whitening and regular dentifrices on orthodontic clear ligature color stability.

PubMed

Oliveira, Adauê S; Kaizer, Marina R; Salgado, Vinícius E; Soldati, Dener C; Silva, Roberta C; Moraes, Rafael R

2015-01-01

This study evaluated the effect of brushing orthodontic clear ligatures with a whitening dentifrice containing a blue pigment (Close Up White Now, Unilever, London, UK) on their color stability, when exposed to a staining agent. Ligatures from 3M Unitek (Monrovia, CA, USA) and Morelli (Sorocaba, SP, Brazil) were tested. Baseline color measurements were performed and nonstained groups (control) were stored in distilled water whereas test groups were exposed for 1 hour daily to red wine. Specimens were brushed daily using regular or whitening dentifrice. Color measurements were repeated after 7, 14, 21, and 28 days using a spectrophotometer based on the CIE L*a*b* system. Decreased luminosity (CIE L*), increased red discoloration (CIE a* axis), and increased yellow discoloration (CIE b* axis) were generally observed for ligatures exposed to the staining agent. Color variation was generally lower in specimens brushed with regular dentifrice, but ligatures brushed with whitening dentifrice were generally less red and less yellow than regular dentifrice. The whitening dentifrice led to blue discoloration trend, with visually detectable differences particularly apparent according to storage condition and ligature brand. The whitening dentifrice containing blue pigment did not improve the ligature color stability, but it decreased yellow discoloration and increased a blue coloration. The use of a whitening dentifrice containing blue pigment during orthodontic treatment might decrease the yellow discoloration of elastic ligatures. © 2015 Wiley Periodicals, Inc.

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Polychromatophilia

MedlinePlus

... than normal with certain dyes. The extra staining is due to too many immature red blood cells (RBCs) called reticulocytes. These cells have a blue-colored center. Increased reticulocytes are the result of ...

New Advances in Molecular Therapy for Muscle Repair after Diseases and Injuries

DTIC Science & Technology

2011-01-01

members of the broader scientific community . Statement of Work...negative for CD34 (1A). Nuclei were stained blue with Dapi. Scale bars, 100 µm. Flow cytometric analysis indicated percentage of cryopreserveded...muscle cells were also cytocentrifuged on glass slides and stained with antibodies to CD56, CD146, UEA-1(2Q, scale bars, 100 µm), and CD56/UEA-1

Computed Tomography-Guided Methylene Blue Labeling Prior to Thoracoscopic Resection of Small Deeply Placed Pulmonary Nodules. Do We Really Need Palpation?

PubMed

Findik, Gokturk; Demiröz, S Mustafa; Apaydın, Selma Mine Kara; Ertürk, Hakan; Biri, Suzan; Incekara, Funda; Aydogdu, Koray; Kaya, Sadi

2017-08-01

Background  Video-assisted thoracic surgery (VATS) is widely used for thoracic surgery operations, and day by day it becomes routine for the excision of undetermined pulmonary nodules. However, it is sometimes hard to reach millimetric nodules through a VATS incision. Therefore, some additional techniques were developed to reach such nodules little in size and which are settled on a challenging localization. In the literature, coils, hook wires, methylene blue, lipidol, and barium staining, and also ultrasound guidance were described for this aim. Herein we discuss our experience with CT-guided methylene blue labeling of small, deeply located pulmonary nodules just before VATS excision. Method  From April 2013 to October 2016, 11 patients with millimetric pulmonary nodules (average 8, 7 mm) were evaluated in our clinic. For all these patients who had strong predisposing factors for malignancy, an 18F-FDG PET-CT scan was also performed. The patients whose nodules were decided to be excised were consulted the radiology clinic. The favorable patients were taken to CT room 2 hours prior to the operation, and CT-guided methylene blue staining were performed under sterile conditions. Results  Mean nodule size of 11 patients was 8.7 mm (6, 2-12). Mean distance from the visceral pleural surface was 12.7 mm (4-29.3). Four of the nodules were located on the left (2 upper lobes, 2 lower lobes), and seven of them were on the right (four lower lobes, two upper lobes, one middle lobe). The maximum standardized uptake values (SUV max) on 18F-FDG PET/CT scan ranged between 0 and 2, 79. Conclusion  CT-guided methylene blue staining of millimetric deeply located pulmonary nodules is a safe and feasible technique that helps surgeon find these undetermined nodules by VATS technique without any need of digital palpation. Georg Thieme Verlag KG Stuttgart · New York.

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital, Argentina.

PubMed

Testardini, Pamela; Vaulet, María Lucía Gallo; Entrocassi, Andrea Carolina; Menghi, Claudia; Eliseht, Martha Cora; Gatta, Claudia; Losada, Mirta; Touzón, María Sol; Corominas, Ana; Vay, Carlos; Tatti, Silvio; Famiglietti, Angela; Fermepin, Marcelo Rodriguez; Perazzi, Beatriz

2016-04-01

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.

Noninvasive intratissue refractive index shaping (IRIS) of the cornea with blue femtosecond laser light.

PubMed

Xu, Lisen; Knox, Wayne H; DeMagistris, Margaret; Wang, Nadan; Huxlin, Krystel R

2011-10-17

To test the feasibility of intratissue refractive index shaping (IRIS) in living corneas by using 400-nm femtosecond (fs) laser pulses (blue-IRIS). To test the hypothesis that the intrinsic two-photon absorption of the cornea allows blue-IRIS to be performed with greater efficacy than when using 800-nm femtosecond laser pulses. Fresh cat corneas were obtained postmortem and cut into six wedges. Blue laser pulses at 400 nm, with 100-fs pulse duration at 80 MHz were used to micromachine phase gratings into each corneal wedge at scanning speeds from 1 to 15 mm/s. Grating lines were 1 μm wide, 5 μm apart, and 150 μm below the anterior corneal surface. Refractive index (RI) changes in micromachined regions were measured immediately by recording the diffraction efficiency of inscribed gratings. Six hours later, the corneas were processed for histology, and TUNEL staining was performed to assess whether blue-IRIS causes cell death. Scanning at 1 and 2 mm/s caused overt corneal damage in the form of bubbles and burns. At faster scanning speeds (5, 10, and 15 mm/s), phase gratings were created in the corneal stroma, which were shown to be pure RI changes ranging from 0.037 to 0.021 in magnitude. The magnitude of RI change was inversely related to scanning speed. TUNEL staining showed cell death only around bubbles and burns. Blue-IRIS can be performed safely and effectively in living cornea. Compared with near-infrared laser pulses, blue-IRIS enhances both achievable RI change and scanning speed without the need to dope the tissue with two-photon sensitizers, increasing the clinical applicability of this technique.

Noninvasive Intratissue Refractive Index Shaping (IRIS) of the Cornea with Blue Femtosecond Laser Light

PubMed Central

Xu, Lisen; Knox, Wayne H.; DeMagistris, Margaret; Wang, Nadan

2011-01-01

Purpose. To test the feasibility of intratissue refractive index shaping (IRIS) in living corneas by using 400-nm femtosecond (fs) laser pulses (blue-IRIS). To test the hypothesis that the intrinsic two-photon absorption of the cornea allows blue-IRIS to be performed with greater efficacy than when using 800-nm femtosecond laser pulses. Methods. Fresh cat corneas were obtained postmortem and cut into six wedges. Blue laser pulses at 400 nm, with 100-fs pulse duration at 80 MHz were used to micromachine phase gratings into each corneal wedge at scanning speeds from 1 to 15 mm/s. Grating lines were 1 μm wide, 5 μm apart, and 150 μm below the anterior corneal surface. Refractive index (RI) changes in micromachined regions were measured immediately by recording the diffraction efficiency of inscribed gratings. Six hours later, the corneas were processed for histology, and TUNEL staining was performed to assess whether blue-IRIS causes cell death. Results. Scanning at 1 and 2 mm/s caused overt corneal damage in the form of bubbles and burns. At faster scanning speeds (5, 10, and 15 mm/s), phase gratings were created in the corneal stroma, which were shown to be pure RI changes ranging from 0.037 to 0.021 in magnitude. The magnitude of RI change was inversely related to scanning speed. TUNEL staining showed cell death only around bubbles and burns. Conclusions. Blue-IRIS can be performed safely and effectively in living cornea. Compared with near-infrared laser pulses, blue-IRIS enhances both achievable RI change and scanning speed without the need to dope the tissue with two-photon sensitizers, increasing the clinical applicability of this technique. PMID:21931133

The nature of iron deposits differs between symptomatic and asymptomatic carotid atherosclerotic plaques

DOE PAGES

Kopriva, David; Kisheev, Anastasye; Meena, Deiter; ...

2015-11-25

Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophagesmore » with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient’s LDL cholesterol (R 2 = 0.39, Significance F = 0.05). Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. Moreover, the abundance of iron in symptomatic plaques is associated with the source patient’s LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin.« less

The Nature of Iron Deposits Differs between Symptomatic and Asymptomatic Carotid Atherosclerotic Plaques

PubMed Central

Kopriva, David; Kisheev, Anastasye; Meena, Deiter; Pelle, Shaneen; Karnitsky, Max; Lavoie, Andrea; Buttigieg, Josef

2015-01-01

Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophages with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient’s LDL cholesterol (R2 = 0.39, Significance F = 0.05). Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. The abundance of iron in symptomatic plaques is associated with the source patient’s LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin. PMID:26606178

Deoxycholic Acid and the Marginal Mandibular Nerve: A Cadaver Study.

PubMed

Blandford, Alexander D; Ansari, Waseem; Young, Jason M; Maley, Bruce; Plesec, Thomas P; Hwang, Catherine J; Perry, Julian D

2018-06-04

One of the rare but serious complications observed with deoxycholic acid administration is damage to the marginal mandibular nerve. In this study, we evaluated if deoxycholic acid directly induces histologic damage to fresh cadaveric marginal mandibular nerve. A segment of marginal mandibular nerve was harvested from 12 hemifaces of 6 fresh cadavers. The nerve specimen was exposed to either 0.9% sterile saline for 24 h, deoxycholic acid (10 mg/ml) for 20 min, or deoxycholic acid (10 mg/ml) for 24 h. The nerve specimens were then fixed in glutaraldehyde for a minimum of 24 h. Toluidine blue stained sections were evaluated for stain intensity using light microscopy and color deconvolution image analysis. Supraplatysmal fat was harvested as a positive control and exposed to the same treatments as the marginal mandibular nerve specimens, then evaluated using transmission electron microscopy. Toluidine blue staining was less in the marginal mandibular nerve exposed to deoxycholic acid when compared to saline. The specimen exposed to deoxycholic acid for 24 h showed less toluidine blue staining than that of the nerve exposed to deoxycholic acid for 20 min. Transmission electron microscopy of submental fat exposed to deoxycholic acid revealed disruption of adipocyte cell membrane integrity and loss of cellular organelles when compared to specimens only exposed to saline. Deoxycholic acid (10 mg/ml) damages the marginal mandibular nerve myelin sheath in fresh human cadaver specimens. Direct deoxycholic acid neurotoxicity may cause marginal mandibular nerve injury clinically. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The nature of iron deposits differs between symptomatic and asymptomatic carotid atherosclerotic plaques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopriva, David; Kisheev, Anastasye; Meena, Deiter

Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophagesmore » with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient’s LDL cholesterol (R 2 = 0.39, Significance F = 0.05). Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. Moreover, the abundance of iron in symptomatic plaques is associated with the source patient’s LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin.« less

Experimental acute thrombotic stroke in baboons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Del Zoppo, G.J.; Copeland, B.R.; Harker, L.A.

1986-11-01

To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9more » cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8.« less

Myelogenous leukemia in a bearded dragon (Acanthodraco vitticeps).

PubMed

Tocidlowski, M E; McNamara, P L; Wojcieszyn, J W

2001-03-01

A 3-yr-old bearded dragon (Acanthodraco vitticeps) presented with lethargy, a swollen right elbow joint, inability to move its rear limbs normally, and marked leukocytosis. The majority of leukocytes were an abnormal mononuclear lymphoid-type cell with a high nuclear to cytoplasmic ratio, a slightly blue cytoplasm, nuclei with coarsely granular chromatin, and some nuclear clefts. Acute leukemia of lymphoid or myeloid origin was tentatively diagnosed. The abnormal mononuclear leukocyte cell population stained positively for the myeloid cytochemical stains: peroxidase, chloroacetate esterase, and L1-calprotectin. The abnormal cell population of the peripheral blood did not stain with the lymphoid cytochemical stains: alpha-naphthyl butyrate esterase, CD3, and CD79a.

Simple and rapid staining for detection of Entamoeba cysts and other protozoans with fluorochromes.

PubMed

Kawamoto, F; Mizuno, S; Fujioka, H; Kumada, N; Sugiyama, E; Takeuchi, T; Kobayashi, S; Iseki, M; Yamada, M; Matsumoto, Y

1987-02-01

Three fluorochromes were applied to stain various parasitic protozoans. By double staining with 4',6-diamidino-2-phenylindole and propidium iodide, differentiation of the nuclei from the cytoplasm can easily be achieved within several seconds. The chromatoid bodies in Entamoeba cysts were stained bright red. Plasmodium yoelii at all stages except late trophozoites and young gametocytes was easily identified. In the oocysts of Cryptosporidium sp., the nuclei and cytoplasm of the sporozoites fluoresced bluish white and red, respectively, whereas the residual body appeared blue or green. The third fluorochrome, Calcofluor white M2R, was suitable for detecting the cysts of Entamoeba spp. and Chilomastix mesnili.

The Rocky Mountain Epidemic of Bark Beetles and Blue Stain Fungi Cause Cascading Effects on Coupled Water, C and N cycles

NASA Astrophysics Data System (ADS)

Ewers, B. E.; Pendall, E.; Norton, U.; Reed, D.; Franks, J.; Aston, T.; Whitehouse, F.; Barnard, H. R.; Brooks, P. D.; Angstmann, J.; Massman, W. J.; Williams, D. G.; Harpold, A. A.; Biederman, J.; Edburg, S. L.; Meddens, A. J.; Gochis, D. J.; Hicke, J. A.

2010-12-01

The ongoing epidemic of bark beetles and their associated xylem blocking blue-stain fungi is unprecedented in Rocky Mountain subalpine forests. As this epidemic continues, we seek to improve our predictive understanding of coupled water, C and N cycles by quantifying how these cycles may become uncoupled in response to the outbreak. Our specific questions are 1) how does the rapid drop in individual tree transpiration impact the temporal and spatial extent of evapotranspiration and 2) how does the subsequent increase in soil moisture and lower C inputs and N uptake impact soil C and N fluxes? We address these questions in two forest ecosystems using eddy covariance, sap flux, leaf gas exchange, plant hydraulic conductance, vegetation characteristics and soil trace gas measurements. We applied two sampling designs 1) subdivide the lodgepole pine forest spatially into varying degrees of bark beetle and blue stain infection and 2) follow the fluxes as the outbreak continues at a point in space encompassing the range of spatial variability in mortality. The first order impact of the bark beetle and blue stain fungi is dramatic in all tree species with a greater than 50% reduction in transpiration per tree within a month of infection. This change occurs even before the characteristic red tinge occurs in the needles or before the sapwood is stained blue. Leaf stomatal conductance declines more than either the biochemical or light harvesting components of photosynthesis immediately after infestation. The annual C sink at the spruce/fir forest has declined from -2.88 to -0.57 Mg C ha-1 yr-1 from 2006 to 2009. Annual evapotranspiration (ET) over the last five years at the spruce/fir forest now has an inverse relationship with precipitation because the last two years have seen a dramatic decrease (from 73 to 59 cm/year) in ET while precipitation has increased (from ~100 to 140 cm/year). Soil moisture in both forests has increased up to 100% within one growing season in lodgepole pine stands with high mortality. At the spruce/fir forest, July soil moisture has increased more than 30% over the last five years. Soil respiration has declined likely from lower labile C inputs while CH4 consumption has decreased due to higher soil moisture. N2O fluxes have increased with mineral N availability. Our results suggest that C, N and water cycles have become decoupled within two years of infestation. However, these soil impacts are not yet as dramatic when scaled up to the eddy covariance footprint which includes uninfested areas. We test these mechanisms at larger scales and investigate C and N cycles as succession occurs using the Community Land Model (CLM).

New observations on endometrial physiology after transcervical injection of methylene blue dye.

PubMed

Marconi, Guillermo; Vilela, Martín; Quintana, Ramiro; Diradourián, Marco; Young, Edgardo; Sueldo, Carlos

2004-12-01

We describe the in vivo features of endometrium stained with methylene blue dye and observed via microhysteroscopy, showing the patterns of endometrial glands and superficial cell changes during the midproliferative, periovulatory, and midluteal phases. These preliminary observations have allowed us to identify a series of changes occurring in the different phases of the ovulatory cycle of potential value in reproductive medicine for specific groups of infertile patients.

The Combination of Trypan Blue and Brilliant Blue G-Assisted Vitrectomy for Macular Pucker: Histopathological Findings.

PubMed

Tognetto, Daniele; De Giacinto, Chiara; D'Aloisio, Rossella; Papagno, Claudia; Pastore, Marco; Zweyer, Marina

2018-01-01

To report on the combined use of trypan blue (TB) and brilliant blue G (BBG) for staining the epiretinal membrane (ERM) and internal limiting membrane (ILM) during vitrectomy and to describe the histopathological findings. 10 surgical specimens were removed from 10 eyes with macular pucker during vitrectomy using a commercially available combination of TB and BBG for ERM and ILM staining and peeling. Specimens were evaluated using light and transmission electron microscopy. In all cases the combination of TB and BBG was useful for identifying and delineating ERM and ILM. No complications related to the use of the dye were observed during or after surgery. Glial cells were present in all specimens. Hyalocytes were observed in 6 cases and myofibroblasts in 3 of them. In 7 cases native vitreous collagen fibrils were found on the ILM, while in 5 specimens newly formed collagen was present. No clinical evidence of toxicity was observed during the 3-month follow-up. The combined use of TB and BBG appeared to be very useful intraoperatively to improve the visualization of ERM and ILM, thus facilitating their complete removal. Anatomical and histopathological findings demonstrated the safety and the efficacy of this vital dye. © 2018 S. Karger AG, Basel.

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

A brief review of other notable protein detection methods on acrylamide gels.

PubMed

Kurien, Biji T; Scofield, R Hal

2012-01-01

Several methods have been described to stain proteins analyzed on acrylamide gels. These include ultrasensitive protein detection in one-dimensional and two-dimensional gel electrophoresis using a fluorescent product from the fungus Epicoccum nigrum; a fluorescence-based Coomassie Blue protein staining; visualization of proteins in acrylamide gels using ultraviolet illumination; fluorescence visualization of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; and increasing the sensitivity four- to sixfold for detecting trace proteins in dye or silver stained polyacrylamide gels using polyethylene glycol 6000. All these methods are reviewed briefly in this chapter.

Comparative evaluation of methylene blue and demeclocycline for enhancing optical contrast of brain neoplasms

NASA Astrophysics Data System (ADS)

Wirth, Dennis J.

Brain tumors cause significant morbidity and mortality even when benign. Completeness of resection of brain tumors has been associated with better quality of life. However, that is often difficult to accomplish. The goal of this study was to evaluate the feasibility of using contrast enhanced multimodal confocal imaging for intraoperative detection of brain neoplasms. Different types of benign and malignant, primary and metastatic brain tumors, stained with Methylene Blue (MB) as a contrast agent, were imaged. MB is a traditional histopathologic stain that absorbs light in the red spectral range and fluoresces in the near infrared. It is FDA-approved for in vivo staining of human skin and breast tissue. Optical images showed good correlation with histopathology, demonstrating the potential of contrast enhanced multimodal confocal imaging for intraoperative detection of brain neoplasms ex vivo. However, the safety of MB for staining human brain in vivo is questionable. Demeclocycline (DMN), an antibiotic of the tetracycline family, has shown to be effective in differentiating normal from cancerous tissue in various organs. DMN is a fluorophore, which absorbs light in the violet spectral range and has a broad emission band covering green and yellow wavelengths. It is commonly used to treat infection and inflammatory disorders, and could provide a safer alternative to MB. To test this hypothesis, fresh excess human brain tissues were bisected and stained with aqueous solutions of either MB or DMN and then imaged. Reflectance and fluorescence images acquired from tissues stained with the two dyes were compared, and correlated with processed H&E histopathology. Comparison showed similar staining patterns and contrast of diagnostic features in glioblastomas, stained using either MB or DMN. The results show potential of both MB and DMN for the intraoperative detection of microscopic nests of brain neoplasms. Further studies will establish safety and efficacy of these agents in vivo.

Synthesis of a novel polyamidoamine dendrimer conjugating with alkali blue as a lymphatic tracer and study on the lymphatic targeting in vivo.

PubMed

Yang, Rui; Xia, Suxia; Ye, Tiantian; Yao, Jianhua; Zhang, Ruizhi; Wang, Shujun; Wang, Siling

2016-09-01

In this study, a novel lymphatic tracer polyamidoamin-alkali blue (PAMAM-AB) was synthesized in order to evaluate the intra-lymphatic targeting ability and lymphatic tropism of PAMAM-AB after subcutaneous administration. UV-Vis, FT-IR, NMR and HPLC characterization were performed to prove the successful synthesis of PAMAM-AB. The calculated AB payload of PAMAM-AB conjugate was seven per dendrimer molecule (27.16% by weight). Hydrolysis stability of PAMAM-AB in vitro was evaluated, which was stable in PBS and human plasma. Lymphatic tracing were studied to determine the blue-stained intensity of PAMAM-AB in right popliteral lymph nodes (PLNs), iliac lymph nodes (ILNs) and para-aortic lymph nodes (PALNs) after subcutaneous administration. The pharmacokinetics and biodistribution of PAMAM-AB in mice were investigated. PLNs, ILNs and PALNs could be obviously blue-stained within 10 min after PAMAM-AB administration, and displayed a more rapid lymphatic absorption, a higher AUC value in lymph nodes and a longer lymph nodes residence time compared with methylene blue solution (MB-S), MB water-in-oil microemulsion (MB-ME), MB multiple microemulsion (MB-MME). Enhanced lymphatic drainage from the injection site and uptake into lymph of PAMAM-AB indicated that PAMAM-AB possesses the double function of lymphatic tracing and lymphatic targeting, and suggested the potential for the development of lymphatic targeting vectors or as a lymphatic tracer in its own right.

Contrasting cellular damage after Blue-IRIS and Femto-LASIK in cat cornea.

PubMed

Wozniak, Kaitlin T; Elkins, Noah; Brooks, Daniel R; Savage, Daniel E; MacRae, Scott; Ellis, Jonathan D; Knox, Wayne H; Huxlin, Krystel R

2017-12-01

Blue-intra-tissue refractive index shaping (Blue-IRIS) is a new approach to laser refractive correction of optical aberrations in the eye, which alters the refractive index of the cornea rather than changing its shape. Before it can be implemented in humans, it is critical to establish whether and to what extent, Blue-IRIS damages the cornea. Here, we contrasted the impact of -1.5 D cylinder refractive corrections inscribed using either Blue-IRIS or femtosecond laser in-situ keratomileusis (femto-LASIK) on corneal cell viability. Blue-IRIS was used to write a -1.5 D cylinder gradient index (GRIN) lens over a 2.5 mm by 2.5 mm area into the mid-stromal region of the cornea in six freshly-enucleated feline eyes. The same correction (-1.5 D cylinder) was inscribed into another four cat eyes using femto-LASIK. Six hours later, all corneas were processed for histology and stained for terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and p-γ-H2AX to label damaged cells. In Blue-IRIS-treated corneas, no tissue was removed and TUNEL-stained cells were confined to the laser focal zone in the stroma. In femto-LASIK, photoablation removed 14 μm of anterior stroma, but in addition, TUNEL-positive cells clustered across the femto-flap, the epithelium at the flap edges and the stroma below the ablation zone. Keratocytes positive for p-γ-H2AX were seen adjacent to all Blue-IRIS focal zones, but were completely absent from femto-LASIK-treated corneas. Unlike femto-LASIK, Blue-IRIS attains refractive correction in the cornea without tissue removal and only causes minimal, localized keratocyte death within the laser focal zones. In addition, Blue-IRIS induced DNA modifications associated with phosphorylation of γ-H2AX in keratocytes adjacent to the laser focal zones. We posit that this p-γ-H2AX response is related to alterations in chromatin structure caused by localized changes in osmolarity, a possible mechanism for the induced refractive index changes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques.

PubMed

Ithoi, Init; Ahmad, Arine-Fadzlun; Mak, J W; Nissapatorn, Veeranoot; Lau, Yee-Ling; Mahmud, Rohela

2011-11-01

Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.

A new procedure for fast soft staining of BN-PAGEs on photosynthetic complexes.

PubMed

Farci, Domenica; Kirkpatrick, Joanna; Piano, Dario

2017-02-01

We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single-step staining-destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photodynamic therapy: a synergy between light and colors

NASA Astrophysics Data System (ADS)

Merigo, Elisabetta; Sozzi, Michele; Ciociola, Tecla; Conti, Stefania; Fornaini, Carlo; Vescovi, Paolo; Selleri, Stefano; Cucinotta, Annamaria

2015-02-01

In this work the application of different laser wavelengths, in combination with different photosensitizing dyes, to bacterial cultures, in liquid or solid mean, has been investigated. Two types of Streptococcus mutans cultures have been used for the experiments, inside agar and saline solution. Three different laser wavelengths have been applied to the bacterial cultures together with a photosensitizing dye: red diode (650 nm) on cultures stained with Toluidine Blue, blueviolet diode (450 nm) on cultures stained with Curcumin and KTP laser (532 nm) on cultures stained with Erythrosine. The choice of the dye has been made considering the color affinity with the used wavelength. Tests without dyes have also been performed. Experimental results show that the maximum inhibition of bacterial growth with the blue laser has been obtained in a saline solution with a growth of 40.77%. While the combination with Curcumin lead to an inhibition growth of about 99.1%, for a laser fluence of 30J/cm2. No inhibition has been observed using the red laser in saline solution without dye, while the combination with Toluidine Blue resulted in a 100% inhibition growth for 20 and 30 J/cm2 fluences. An inhibition growth of just 16.26% has been obtained with the use of KTP laser in saline solution without dye. The use of Erythrosine had the effect of a complete inhibition growth. From the obtained results it is possible to observe that the combination of laser wavelength with a particular photosensitizing dye can dramatically increase the bacterial growth.

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin.

PubMed

Sullivan-Brown, Jessica; Bisher, Margaret E; Burdine, Rebecca D

2011-01-01

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

Effect of blue light radiation on curcumin-induced cell death of breast cancer cells

NASA Astrophysics Data System (ADS)

Zeng, X. B.; Leung, A. W. N.; Xia, X. S.; Yu, H. P.; Bai, D. Q.; Xiang, J. Y.; Jiang, Y.; Xu, C. S.

2010-06-01

In the present study, we have successfully set up a novel blue light source with the power density of 9 mW/cm2 and the wavelength of 435.8 nm and then the novel light source was used to investigate the effect of light radiation on curcumin-induced cell death. The cytotoxicity was investigated 24 h after the treatment of curcumin and blue light radiation together using MTT reduction assay. Nuclear chromatin was observed using a fluorescent microscopy with Hoechst33258 staining. The results showed blue light radiation could significantly enhance the cytotoxicity of curcumin on the MCF-7 cells and apoptosis induction. These findings demonstrated that blue light radiation could enhance curcumin-induced cell death of breast cancer cells, suggesting light radiation may be an efficient enhancer of curcumin in the management of breast cancer.

Paranuclear blue inclusions in metastatic undifferentiated small cell carcinoma in the bone marrow.

PubMed

Wittchow, R; Laszewski, M; Walker, W; Dick, F

1992-09-01

Paranuclear blue inclusions (PBIs) are frequently identified within metastatic undifferentiated small cell carcinoma (SCC) cells on air-dried bone marrow aspirates stained with Wright's stain. To determine the sensitivity and specificity of this finding, 116 bone marrow aspirates containing metastatic neoplasms were evaluated for the presence and frequency of PBIs. Bone marrow specimens included 47 cases of metastatic SCC of the lung, 13 cases of large cell lymphoma, 19 cases of neuroblastoma, five cases of small, noncleaved cell lymphoma, seven cases of rhabdomyosarcoma, three cases of Ewing's sarcoma, three cases of other sarcomas, and 19 cases of non-small cell carcinoma (adenocarcinoma). PBIs were identified in 40 of 47 (85%) cases of SCC and their frequency varied from 0 to 24% of tumor cells among different cases. In approximately half the cases of SCC, PBIs were identified in 1 to 4% tumor cells; and in eight cases, PBIs were present in 5% or more of tumor cells. PBIs were also identified in two of seven (29%) cases of rhabdomyosarcoma and one case of malignant peripheral nerve sheath tumor, but they were not seen in Ewing's sarcoma, small non-cleaved cell lymphoma, large cell lymphoma, neuroblastoma, or non-small cell carcinoma. In addition, PBIs were not seen in alcohol-fixed, Papanicolaou-stained cytology specimens containing SCC. Ultrastructurally, PBIs may represent phagocytized nuclear/cellular material. PBIs are a feature of small cell carcinoma on air-dried, cytologic material stained with Romanowsky type stains. Their presence may provide diagnostic information with regard to the differential diagnosis of metastatic SCC in the bone marrow. Future studies evaluating non-bone marrow Wright's stained fine-needle aspiration specimens are needed to determine if PBIs are useful in distinguishing SCC from other poorly differentiated tumors in the cytology laboratory.

The cytochemical and ultrastructural characteristics of phagocytes in the Pacific oyster Crassostrea gigas.

PubMed

Jiang, Shuai; Jia, Zhihao; Xin, Lusheng; Sun, Ying; Zhang, Ran; Wang, Weilin; Wang, Lingling; Song, Linsheng

2016-08-01

Phagocytes have been proved to play vital roles in the innate immune response. However, the cellular characteristics of phagocytes in invertebrates, especially in molluscs, remain largely unknown. In the present study, fluorescence activated cell sorting (FACS) was employed to sort the phagocytes from the non-phagocytic haemocytes of the Pacific oyster Crassostrea gigas. The cytochemical staining analysis revealed that phagocytes were positive staining for α-naphthyl acetate esterase and myeloperoxidase, while negative staining for toluidine blue and periodic acid-Schiff. The non-phagocytic haemocytes exhibited positive staining for periodic acid-Schiff, weak positive staining for toluidine blue, but negative staining for α-naphthyl acetate esterase and myeloperoxidase. In addition, phagocytes exhibited ultrastructural cellular features similar to those of macrophages, with large cell diameter, rough cell membrane and extended pseudopodia revealed by the scanning electron microscopy, while the non-phagocytic haemocytes exhibited small cell diameter, smooth cell surface and round spherical shape. Transmission electron microscopy further demonstrated that phagocytes were abundant of cytoplasmic bodies and mitochondria, while non-phagocytic haemocytes were characterized as the comparatively large cell nucleus with contorted and condensed heterochromatin adherent to the nuclear envelope. Moreover, compared with non-phagocytic haemocytes, phagocytes exhibited significantly higher levels of intracellular cytokines, including tumor necrosis factor, interferon-like protein and interleukin-17, and significantly higher abundance of lysosome and reactive oxygen species, which were of great importance to the activation of immune response and pathogen clearance. Taken together, these findings revealed the different cytochemical and ultrastructural features between phagocytes and non-phagocytic haemocytes in C. gigas, which would provide an important clue to investigate the mechanism of phagocytosis underlying the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydroxy-Al and cell-surface negativity are responsible for the enhanced sensitivity of Rhodotorula taiwanensis to aluminum by increased medium pH.

PubMed

Zhao, Xue Qiang; Bao, Xue Min; Wang, Chao; Xiao, Zuo Yi; Hu, Zhen Min; Zheng, Chun Li; Shen, Ren Fang

2017-10-01

Aluminum (Al) is ubiquitous and toxic to microbes. High Al 3+ concentration and low pH are two key factors responsible for Al toxicity, but our present results contradict this idea. Here, an Al-tolerant yeast strain Rhodotorula taiwanensis RS1 was incubated in glucose media containing Al with a continuous pH gradient from pH 3.1-4.2. The cells became more sensitive to Al and accumulated more Al when pH increased. Calculations using an electrostatic model Speciation Gouy Chapman Stern indicated that, the increased Al sensitivity of cells was associated with AlOH 2+ and Al(OH) 2 + rather than Al 3+ . The alcian blue (a positively charged dye) adsorption and zeta potential determination of cell surface indicated that, higher pH than 3.1 increased the negative charge and Al adsorption at the cell surface. Taken together, the enhanced sensitivity of R. taiwanensis RS1 to Al from pH 3.1-4.2 was associated with increased hydroxy-Al and cell-surface negativity.

Morphology and evolutionary implications of the annual cycle of secretion and sperm storage in spermathecae of the salamander Ambystoma opacum (Amphibia: Ambystomatidae).

PubMed

Sever, David M; Krenz, John D; Johnson, Kristin M; Rania, Lisa C

1995-01-01

Females of the marbled salamander, Ambystoma opacum, store sperm in exocrine glands called spermathecae in the roof of the cloaca. Eggs are fertilized by sperm released from the spermathecae during oviposition. Some sperm remain in the spermathecae following oviposition, but these sperm degenerate within a month and none persists more than 6 mo after oviposition. Thus, sperm storage between successive breeding seasons does not occur. Apical secretory vaculoes are abundant during the fall mating season and contain a substance that is alcian blue+ at pH 2.5. Production of secretory vacuoles decreases markedly after oviposition, and the glands are inactive by the summer months. Ambystoma opacum is a terrestrial breeder, and some mating occurs prior to arrival at pond basins where oviposition occurs. Mating prior to arrival at the ovipository site may prolong the breeding season, leading to fitness implications for both males and females. Females have opportunities for more matings, and the possibilities for sperm competition in the spermathecae are enhanced. © 1995 Wiley-Liss, Inc. Copyright © 1995 Wiley-Liss, Inc.

Organic or organometallic template mediated clay synthesis

DOEpatents

Gregar, Kathleen C.; Winans, Randall E.; Botto, Robert E.

1994-01-01

A method for incorporating diverse Varieties of intercalants or templates directly during hydrothermal synthesis of clays such as hectorite or montmorillonite-type layer-silicate clays. For a hectorite layer-silicate clay, refluxing a gel of silica sol, magnesium hydroxide sol and lithium fluoride for two days in the presence of an organic or organometallic intercalant or template results in crystalline products containing either (a) organic dye molecules such as ethyl violet and methyl green, (b) dye molecules such as alcian blue that are based on a Cu(II)-phthalocyannine complex, or (c) transition metal complexes such as Ru(II)phenanthroline and Co(III)sepulchrate or (d) water-soluble porphyrins and metalloporphyrins. Montmorillonite-type clays are made by the method taught by U.S. Pat. No. 3,887,454 issued to Hickson, Jun. 13, 1975; however, a variety of intercalants or templates may be introduced. The intercalants or templates should have (i) water-solubility, (ii) positive charge, and (iii) thermal stability under moderately basic (pH 9-10) aqueous reflux conditions or hydrothermal pressurized conditions for the montmorillonite-type clays.

Organic or organometallic template mediated clay synthesis

DOEpatents

Gregar, K.C.; Winans, R.E.; Botto, R.E.

1994-05-03

A method is described for incorporating diverse varieties of intercalates or templates directly during hydrothermal synthesis of clays such as hectorite or montmorillonite-type layer-silicate clays. For a hectorite layer-silicate clay, refluxing a gel of silica sol, magnesium hydroxide sol and lithium fluoride for two days in the presence of an organic or organometallic intercalate or template results in crystalline products containing either (a) organic dye molecules such as ethyl violet and methyl green, (b) dye molecules such as alcian blue that are based on a Cu(II)-phthalocyannine complex, or (c) transition metal complexes such as Ru(II)phenanthroline and Co(III)sepulchrate or (d) water-soluble porphyrins and metalloporphyrins. Montmorillonite-type clays are made by the method taught by U.S. Pat. No. 3,887,454 issued to Hickson, Jun. 13, 1975; however, a variety of intercalates or templates may be introduced. The intercalates or templates should have (i) water-solubility, (ii) positive charge, and (iii) thermal stability under moderately basic (pH 9-10) aqueous reflux conditions or hydrothermal pressurized conditions for the montmorillonite-type clays. 22 figures.

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus.

PubMed

Prusch, R D

1986-08-01

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca2+ in the external medium. Increasing external Ca2+ to approximately 10(-4) M increases pinocytotic intensity, while increases in Ca2+ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca2+ at the surface of Amoeba proteus was therefore examined. Alcian blue and Na+, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca2+ in the range of 3 X 10(-5) to 1.5 X 10(-4) M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca2+ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity.

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in biofilms by pulsed ultraviolet light.

PubMed

Montgomery, Nedra L; Banerjee, Pratik

2015-06-10

The inactivation of biofilms formed by pathogenic bacteria on ready-to-eat and minimally processed fruits and vegetables by nonthermal processing methods is critical to ensure food safety. Pulsed ultraviolet (PUV) light has shown promise in the surface decontamination of liquid, powdered, and solid foods. In this study, the antimicrobial efficacy of PUV light treatment on nascent biofilms formed by Escherichia coli O157:H7 and Listeria monocytogenes on the surfaces of food packaging materials, such as low-density polyethylene (LDPE), and fresh produce, such as lettuce (Lactuca sativa) leaves, was investigated. The formation of biofilms on Romaine lettuce leaves and LDPE films was confirmed by crystal violet and Alcian blue staining methods. Inactivation of cells in the biofilm was determined by standard plating procedures, and by a luminescence-based bacterial cell viability assay. Upon PUV treatment of 10 s at two different light source to sample distances (4.5 and 8.8 cm), viable cell counts of L. monocytogenes and E. coli O157:H7 in biofilms on the lettuce surface were reduced by 0.6-2.2 log CFU mL(-1) and 1.1-3.8 log CFU mL(-1), respectively. On the LDPE surface, the efficiency of inactivation of biofilm-encased cells was slightly higher. The maximum values for microbial reduction on LDPE were 2.7 log CFU mL(-1) and 3.9 log CFU mL(-1) for L. monocytogenes and E. coli O157:H7, respectively. Increasing the duration of PUV light exposure resulted in a significant (P < 0.05) reduction in biofilm formation by both organisms. The results also revealed that PUV treatment was more effective at reducing E. coli biofilms compared with Listeria biofilms. A moderate increase in temperature (~7-15°C) was observed for both test materials. PUV is an effective nonthermal intervention method for surface decontamination of E. coli O157:H7 and L. monocytogenes on fresh produce and packaging materials.

Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells

PubMed Central

de Vries, Stefan A.H.; Potier, Esther; van Doeselaar, Marina; Meij, Björn P.; Tryfonidou, Marianna A.

2015-01-01

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed. PMID:25370929

Lubiprostone ameliorates the cystic fibrosis mouse intestinal phenotype.

PubMed

De Lisle, Robert C; Mueller, Racquel; Roach, Eileen

2010-09-15

Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl- channel, would improve the intestinal phenotype in CF mice. Cftr(tm1UNC) (CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR. Crypt width in control CF mice was 700% that of WT mice (P < 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P = 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P = 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P = 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (P < 0.001) and CF mice (P < 0.001). Lubiprostone enhanced small intestinal transit in WT mice (P = 0.024) but not in CF mice (P = 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels. These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.

Lubiprostone ameliorates the cystic fibrosis mouse intestinal phenotype

PubMed Central

2010-01-01

Background Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl- channel, would improve the intestinal phenotype in CF mice. Methods Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR. Results Crypt width in control CF mice was 700% that of WT mice (P < 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P = 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P = 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P = 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (P < 0.001) and CF mice (P < 0.001). Lubiprostone enhanced small intestinal transit in WT mice (P = 0.024) but not in CF mice (P = 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels. Conclusions These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. PMID:20843337

The septal body revisited.

PubMed

Elwany, S; Salam, S A; Soliman, A; Medanni, A; Talaat, E

2009-03-01

The term septal body refers to a thickened area of the nasal septum which is located superior to the inferior turbinate and anterior to the middle turbinate. Despite its important role in changing nasal airflow resistance, it has received little attention. Clinically, a well developed septal body may be misdiagnosed as high septal deviation. The aim of the present study was to reassess the histological characteristics of the septal body mucosa and the morphometric differences between it and the adjacent septal mucosa. This information was then used to determine the exact location and surface area of the septal body. The study was performed on 30 cadaveric specimens (60 sides). Serial numbered sections of the whole septal mucosa were stained with haematoxylin and eosin as well as periodic acid Schiff - Alcian blue. Morphometric analysis was performed to determine the histological differences between the septal body mucosa, the anterior septal mucosa and the inferior septal mucosa. The precise boundaries of the septal body area were then defined in a manner similar to the Mohs micrographic surgical technique. The histological characteristics of the septal body mucosa included thick (more than 60 microm), pseudostratified, ciliated respiratory epithelium with goblet cells, abundant seromucinous glands and many blood sinusoids. Morphometric analysis showed that the septal body mucosa had thicker epithelium and more glandular acini and blood sinusoids than the rest of the septal mucosa. Mapping of the septal body area showed that its anterior end was 2.2 +/- 0.3 cm (mean +/- standard deviation) behind the caudal edge of the septal cartilage, and its inferior border was 1.1 +/- 0.2 cm above the floor of the nose. The mean horizontal diameter of the septal body was 2.0 +/- 0.15 cm, and the mean vertical diameter was 1.5 +/- 0.11 cm. The present study determined the morphometric characteristics of the septal body as well as its location and surface area. The intimate relationship of the septal body to the internal nasal valve and the histological characteristics of its mucosa should stimulate research into its potential role in modifying nasal airflow pattern and resistance, and its role in changing the humidity and temperature of the inspiratory air stream.

Short-term in vivo evaluation of novel vital dyes for intraocular surgery.

PubMed

Haritoglou, Christos; Tadayoni, Ramin; May, Christian A; Gass, Carolin A; Freyer, Wolfgang; Priglinger, Siegfried G; Kampik, Anselm

2006-01-01

To evaluate the staining characteristics and safety of potential new dyes for intraocular surgery in porcine eyes. Four dyes in different solutions (light green SF yellowish [LGSF]: 2%; copper(II) phthalocyanine-tetrasulfonic acid [E68]: 2% and 0.5%; bromophenol blue [BPB]: 2%, 1%, and 0.2%; and Chicago blue [CB]: 2% and 0.5%) were included in this investigation. All dyes were dissolved and diluted using balanced salt solution (BSS plus; Alcon Laboratories, Inc., Fort Worth, TX). After triamcinolone-assisted vitrectomy on 10 porcine eyes in vivo, the dyes were first injected into the air-filled vitreous cavity. After 1 minute, the dye was removed by irrigation with BSS, and the staining effect was graded by two examiners. After vitrectomy, the same dyes and concentrations were injected in the air-filled anterior chamber to stain the lens capsule of the same eye. After surgery, the eyes were enucleated and underwent fixation for light and electron microscopy. The animals were killed by injection of pentobarbital (50 mg/kg). For controls, each BSS plus alone and indocyanine green 0.5% were applied in one eye. On the retinal surface, bright staining of the retinal surface was seen after application of BPB 2% and 1%. The staining effect was less pronounced but still very good using E68 2%, and CB 2% and weak using BPB 0.2%, E68 0.5% and CB 0.5% as well as indocyanine green 0.5%. No staining of the retinal surface but of the vitreous was seen after application of LGSF 2%. The lens capsule stained very well with E68 2%, CB 2% and 0.5%, and BPB 2%, 1%, and 0.2% but not with LGSF. No histologic abnormalities were seen after the application in any eye after dye injection. No dye-related complications occurred during surgery. In this study, we identified three dyes with satisfying staining characteristics in both anterior and posterior segments. Because BPB stained the retinal surface and lens capsule at a low concentration (0.2%) with no signs of toxicity, this dye seems to be the most promising candidate for application in humans.

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

PubMed

Mohebali, Mehdi; Zarei, Zabiholah; Khanaliha, Khadijeh; Kia, Eshrat Beigom; Motavalli-Haghi, Afsaneh; Davoodi, Jaber; Rezaeian, Tahereh; Tarighi, Fathemeh; Rezaeian, Mostafa

2017-01-01

Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, C ricetulus migratorius . This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%) , Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran

PubMed Central

MOHEBALI, Mehdi; ZAREI, Zabiholah; Khanaliha, Khadijeh; KIA, Eshrat Beigom; MOTAVALLI-HAGHI, Afsaneh; DAVOODI, Jaber; REZAEIAN, Tahereh; TARIGHI, Fathemeh; REZAEIAN, Mostafa

2017-01-01

Background: Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius. Methods: This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. Results: About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. Conclusion: Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans. PMID:28979348

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

PubMed

Lee, So-Yeon; Ha, Eun-Ju; Woo, Seung-Kyun; Lee, So-Min; Lim, Kyung-Hee; Eom, Yong-Bin

2017-07-01

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining.

PubMed

Fathi, Mohamed; Ashry, Mohamed; Salama, Ali; Badr, Magdy R

2017-08-01

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

How Long Will I Be Blue? Prolonged Skin Staining Following Sentinel Lymph Node Biopsy Using Intradermal Patent Blue Dye

PubMed Central

Gumus, Metehan; Gumus, Hatice; Jones, Sue E; Jones, Peter A; Sever, Ali R; Weeks, Jennifer

2013-01-01

Summary Background Blue dye used for sentinel lymph node biopsy (SLNB) in breast cancer patients may cause prolonged skin discoloration at the site of injection. The aim of this study was to assess the duration of such skin discoloration. Patients and Methods 236 consecutive patients who had undergone breast conserving surgery and SLNB for breast cancer were reviewed prospectively from January 2007 to December 2009. Results Of the 236 patients, 2 had undergone bilateral surgery, and 41 had been examined in consecutive yearly reviews. Blue discoloration remained visible at the injection site after 12, 24, and > 36 months in 36.5, 23.6, and 8.6% of the patients, respectively. Conclusion The use of patent blue for identification of the sentinel lymph node in patients undergoing breast cancer surgery may result in prolonged discoloration of the skin at the injection site. PMID:24415970

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

NASA Astrophysics Data System (ADS)

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; Dimarzio, Charles; Rajadhyaksha, Milind

2011-07-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

A new fluorescent test for cell vitality using calcofluor white M2R.

PubMed

Fischer, J M; Peterson, C A; Bols, N C

1985-03-01

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.

[Seven cases of parathyroidectomy for secondary hyperparathyroidism using methylene blue: suggestion for the method of methylene blue infusion].

PubMed

Kadoya, Tatsuo; Kinoshita, Yuki; Shiraishi, Munehiro; Uehara, Hirofumi; Yamamoto, Toshinori; Suetsugu, Keiko

2014-08-01

Intraoperative staining of the parathyroid glands with intravenously administered methylene blue is well described and has been demonstrated as an effective and safe method to facilitate parathyroidectomy. However, there have been several literatures of the development of postoperative neurological toxicity in patients who received methylene blue infusion during parathyroidectomy. We report the method of methylene blue infusion during parathyroidectomy at our institution. Seven adult patients who had undergone parathyroidectomy for secondary hyperparathyroidism associated with chronic renal failure were included in this study. Methylene blue was administered at a constant rate of 4 mg x kg(-1) x hr(-1) with a 1% solution just before the start of operation. The infusion was stopped after the first parathyroid gland was identified. The mean dose of methylene blue used was 2.2 +/- 0.8 mg x kg(-1). Consequently, the dose of methylene blue by this method could be decreased to less than half of the previously administered dose (6 mg x kg(-1)) at our institution. The dose of methylene blue used should be kept to the minimum required to identify the parathyroid glands in each case.

A study of hydrogen peroxide chemistry and photochemistry in tea stain solution with relevance to clinical tooth whitening.

PubMed

Young, Nigel; Fairley, Peter; Mohan, Veena; Jumeaux, Coline

2012-12-01

Tooth whitening using hydrogen peroxide is a complex process, and there is still some controversy about the roles of pH, temperature, chemical activators, and the use of light irradiation. In this work the basic interactions between whitening agents and stain molecules are studied in simple solutions, thus avoiding the physics of diffusion and light penetration in the tooth to give clarity on the basic chemistry which is occurring. The absorbance of tea stain solution at 450 nm was measured over a period of 40 min, with various compositions of whitening agent added (including hydrogen peroxide, ferrous gluconate and potassium hydroxide) and at the same time the samples were subjected to blue light (465 nm) or infra-red light (850 nm) irradiation, or alternatively they were heated to 37°C. It is shown that the reaction rates between chromogens in the tea solution and hydrogen peroxide can be accelerated significantly using ferrous gluconate activator and blue light irradiation. Infra red irradiation does not increase the reaction rate through photochemistry, it serves only to increase the temperature. Raising the temperature leads to inefficiency through the acceleration of exothermic decomposition reactions which produce only water and oxygen. By carrying out work in simple solution it was possible to show that ferrous activators and blue light irradiation significantly enhance the whitening process, whereas infra red irradiation has no significant effect over heating. The importance of controlling the pH within the tooth structure during whitening is also demonstrated. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inflammation-associated gene transcription and expression in mouse lungs induced by low molecular weight compounds from fungi from the built environment.

PubMed

Miller, J D; Sun, M; Gilyan, A; Roy, J; Rand, T G

2010-01-05

Few metabolites from fungi found indoors have been tested for inflammatory mediators endpoints in primary cultures of alveolar macrophages or in vivo. In this study, mice were intratracheally instilled with a single dose comprising 4x10(-5)moletoxin/kg lung wt dose of either atranone C, brevianamide, cladosporin, mycophenolic acid, neoechinulin A & B, sterigmatocystin or TMC-120A. These toxins are from fungi common on damp building materials. The dose used was comparable to the estimated doses of possible human exposure. Hematoxylin and eosin (H&E) histology and Alcian Blue/Periodic Acid Schiff (AB/PAS) histochemistry were used to evaluate lungs for time course (4h and 12h post-exposure (PE)) inflammatory and toxic changes. Reverse-transcription (RT)-PCR based arrays were also employed to evaluate time course inflammation-associated gene transcription in lung tissues of the different toxins. Immunohistochemistry (IHC) was used to probe MIP-2 and Tnf-alpha protein expression in treatment lungs to determine whether responses correspond with gene transcription data. Both histology and histochemistry revealed that toxin exposed lungs at 12h PE showed evidence of inflammation. H&E revealed that bronchioli were lined with irregularly thickened and sometimes sloughing epithelium and bronchiolar spaces supported infiltration of leukocytes, cellular and mucus-like debris while alveolar spaces supported swollen macrophages and modest amorphous debris accumulations. All toxin-instilled lungs exhibited copious mucus production and alveolar macrophages with red stained cytoplasm on bronchiolar surfaces, especially at 12h PE. Array analysis of 83 inflammation-associated genes extracted from lung tissue demonstrated a number of patterns, compared to controls. 82 genes assayed at 4h PE and 75 genes at 12h PE were significantly altered (p< or =0.05; >or =1.5-fold or < or =-1.5-fold change) in the different treatment animal groups. Expression of transcriptionally regulated genes was confirmed using immunohistochemistry that demonstrated MIP-2 and Tnf-alpha staining in respiratory bronchiolar epithelia, alveolar macrophages and alveolar type II cells. The transcriptional regulation in these genes in the treatment groups suggests that they may serve central roles in the immunomodulation of toxin-induced pro-inflammatory lung responses. Hierarchical cluster analysis revealed significant patterns of gene transcription linking the response of the toxins at equimolar doses in three groups: (1) brevianamide, mycophenolic acid and neoechinulin B, (2) neoechinulin A and sterigmatocystin, and (3) cladosporin, atranone C and TMC-120. The results further confirm the inflammatory nature of metabolites/toxins from such fungi can contribute to the development of non-allergenic respiratory health effects.

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

Survival of Staphylococcus pseudintermedius in modified Romanowsky staining solutions.

PubMed

Misan, Angus; Chan, Wei Yee; Trott, Darren; Hill, Peter B

2017-08-01

Stains that are used regularly for patient-side diagnosis to rapidly identify bacterial and fungal infections could become contaminated by common pathogens, such as Staphylococcus pseudintermedius, during slide immersion. To determine whether the inoculation of S. pseudintermedius into modified Romanowsky type stains (Quick Dip ® ) results in viable bacterial contamination and whether this is influenced by the addition of organic debris (canine hair and skin). A clinical isolate of S. pseudintermedius was inoculated into clean and organically contaminated Quick Dip ® solutions (methanol fixative, eosin, methylene blue), and positive (broth) and negative (bleach) controls. Each solution was tested for the presence of viable bacteria by counting the number of colony forming units (CFU/mL) at various time points. Solutions also were examined under high power microscopy to count the number of visible bacteria at each time point. Staphylococcus pseudintermedius was able to survive in the clean and contaminated Quick Dip ® stains for at least one hour, but by 24 h no viable bacteria remained. Survival of the bacteria was not supported in the fixative at any time point. Staphylococcus pseudintermedius remained visible under high power microscopy for up to 2 weeks in all organically contaminated solutions of the Quick Dip ® set. Staphylococcus pseudintermedius only remains viable in eosin and methylene blue for short periods of time, but the prolonged visibility of dead organisms could theoretically lead to the misdiagnosis of cytology samples. © 2017 ESVD and ACVD.

Toluidine blue aids in detection of dysplasia and carcinoma in suspicious oral lesions.

PubMed

Chainani-Wu, N; Madden, E; Cox, D; Sroussi, H; Epstein, J; Silverman, S

2015-10-01

Accurate clinical identification of 'higher-risk' oral premalignant lesions or 'higher-risk' areas within lesions is important. Assessment methods that predict their presence have great utility. A cross-sectional, observational study enrolled a consecutive sample of consenting patients diagnosed with oral leukoplakia, erythroleukoplakia, or erythroplakia. Medical history, visual oral examination, ViziLite(®) examination, toluidine blue staining (TBlue(®) ), and finally a biopsy were completed in a single clinic visit. Seventy-seven of 100 examined lesions in 43 patients were biopsied. Sensitivity, specificity, and positive and negative predictive values were computed for visual examination, ViziLite(®) , and TBlue(®) using biopsy results as the gold standard. The sensitivity of TBlue(®) in detecting high-risk lesions (carcinoma in situ or carcinoma) was 94 (71-100, P < 0.0003) and specificity 45 (32-58, P < 0.53), while for carcinoma, sensitivity was 100 (54-100, P < 0.032) and specificity 39 (28-52, P < 0.097). The results of ViziLite(®) testing either by itself or in combination with the information from toluidine blue testing revealed low sensitivity for the detection of high-risk lesions. Clinical examination of leukoplakia, erythroplakia, or erythroleukoplakia lesions combined with toluidine blue staining may aid in the identification of severe dysplasia (carcinoma in situ) or carcinoma. This may help in determining whether, when, and where (the site within a lesion) a biopsy should be taken. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Limited effectiveness of patent blue dye in addition to isotope scanning for identification of sentinel lymph nodes: Cross-sectional real-life study in 1024 breast cancer patients.

PubMed

Rauch, Philippe; Merlin, Jean-Louis; Leufflen, Lea; Salleron, Julia; Harlé, Alexandre; Olivier, Pierre; Marchal, Frédéric

2016-09-01

Although morbidity is reduced when sentinel lymph node (SLN) biopsy is performed with dual isotopic and blue dye identification, the effectiveness of adding blue dye to radioisotope remains debated because side effects including anaphylactic reactions. Using data from a prospectively maintained database, 1884 lymph node-negative breast cancer patients who underwent partial mastectomy with SLN mapping by a dual-tracer using patent blue dye (PBD) and radioisotope were retrospectively studied between January 2000 and July 2013. Patients with tumors <3 cm and with >1 node detected by one of the two techniques (N = 1024) were included in this real-life cross-sectional study. Among the 1024 patients, 274 had positive SLN detected by isotopic and/or PBD staining. Only 4 patients having no detectable radioactivity in the axilla had SLN identified only by PBD staining (blue-only) while 26 patients had SLN only identified by isotopic detection (hot-only) illustrating failure rates of 9.5% (26/274) and 1.5% (4/274), respectively. Among these four patients, two had negative lymphoscintigraphy. Therefore, the contribution of PBD to metastatic nodes identification was relevant for only 2/274 patients (0.8%). Three patients (0.3%) had an allergic reaction with PBD, and anaphylactic shock occurred in two cases (0.2%). The added-value of PBD to reduce the false-negative rate of SLN mapping is only limited to the rare cases in which no radioactivity is detectable in the axilla (<1%). When a radioisotope mapping agent is available, the use of PBD should be avoided, because it can induce anaphylaxis. Copyright © 2016 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

Regional differences in the blood-brain-barrier of the subfornical organs of rats and ducks (Anas platyrhynchos).

PubMed

Schmid, H A

1995-01-01

Recently published electrophysiological data investigated the effect of blood borne and brain intrinsic substances on the activity of neurons in the duck subfornical organ (SFO). This study defines histologically the region in the duck SFO, where blood borne substances can possibly influence neuronal activity. Intravenous injection of Evans blue, a dye which labels brain structures devoid of a blood brain barrier (BBB), resulted in diffuse labelling of the duck SFO from the anterior commissure to the end of the organ in rostrocaudal extension. In addition, specifically labelled neurons could be observed just rostral to the diffuse Evans blue labelling and in an area dorsomedial to the large central blood vessel. The majority of the somata of these heavily stained neurons were located inside the BBB, whereas in the areas with diffuse Evans blue labelling, thus being outside the BBB, labelled cells were rarely observed. Intravenous injection of Evans blue in rats resulted similarly in diffuse labelling of the parenchyma of the medial and caudal part of the SFO, with only a few, but heavily stained cells with fusiform somata. The rostral region of the rat SFO, which is known to have a functional BBB, shows hardly any diffuse labelling, but there the majority of neurons show strong Evans blue fluorescence. It is concluded that the heavily labelled somata inside the BBB have axonal or dendritic projections to BBB-free areas, where they can take up the dye. This study gives a functional description of the extension of the SFO areas without a BBB of rats and ducks. It is concluded that blood borne agents can affect those SFO neurons which have their somata located outside the BBB as well as those located inside the BBB which have terminals projecting to BBB free regions.

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

PubMed Central

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

2011-01-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology. PMID:21806269

Post-treatment Vascular Leakage and Inflammatory Responses around Brain Cysts in Porcine Neurocysticercosis

PubMed Central

Mahanty, Siddhartha; Orrego, Miguel Angel; Mayta, Holger; Marzal, Miguel; Cangalaya, Carla; Paredes, Adriana; Gonzales-Gustavson, Eloy; Arroyo, Gianfranco; Gonzalez, Armando E.; Guerra-Giraldez, Cristina; García, Hector H.; Nash, Theodore E.

2015-01-01

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation. PMID:25774662

Quantitative gel electrophoresis: new records in precision by elaborated staining and detection protocols.

PubMed

Deng, Xi; Schröder, Simone; Redweik, Sabine; Wätzig, Hermann

2011-06-01

Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

PubMed Central

Ramírez-Melgar, Carmen; Gómez-Priego, Alberto

2007-01-01

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 × magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures. PMID:17374981

Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern.

PubMed

Mohapatra, Sushil K; Sandhu, Anjit; Neerukattu, Venkata S; Singh, Karn P; Selokar, Naresh L; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

2015-04-01

We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB-). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB- oocytes (43.41 ± 2.54 vs. 22.74 ± 1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB- blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB- blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB- blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB- blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced by In Vitro Fertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

PubMed Central

Mohapatra, Sushil K.; Sandhu, Anjit; Neerukattu, Venkata S.; Singh, Karn P.; Selokar, Naresh L.; Singla, Suresh K.; Chauhan, Manmohan S.; Manik, Radhey S.

2015-01-01

Abstract We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB−). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB− oocytes (43.41±2.54 vs. 22.74±1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB− blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB− blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB− blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB− blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC. PMID:25826727

Sentinel lymph node detection following the hysteroscopic peritumoural injection of 99mTc-labelled albumin nanocolloid in endometrial cancer.

PubMed

Maccauro, Marco; Lucignani, Giovanni; Aliberti, Gianluca; Villano, Carlo; Castellani, Maria Rita; Solima, Eugenio; Bombardieri, Emilio

2005-05-01

The purpose of this study was to assess the feasibility of sentinel lymph node (SLN) detection in endometrial cancer patients with a dual-tracer procedure after hysteroscopic peritumoural injection. Twenty-six women with previously untreated endometrial adenocarcinoma underwent the hysteroscopic injection of 111 MBq 99mTc-Nanocoll and blue dye administered subendometrially around the lesion. On the same day, all 26 patients underwent lymphoscintigraphy, followed 3-4 h later by hysterotomy with bilateral salpingo-oophorectomy and pelvic lymphadenectomy. Para-aortic lymphadenectomy was also performed in cases of either serous or papillary carcinoma (n=7/26). All SLNs were removed and examined with haematoxylin and eosin staining and immunohistochemical techniques. The procedure was well tolerated by patients, only two experiencing transient vagal symptoms. The sensitivity of this technique for correct identification of SLNs was 100%. Lymph node metastases were found in 4 out of the 26 patients (15%), bilaterally in the external iliac region (n=1), unilaterally in the external iliac region (n=1), unilaterally in the common iliac region (n=1) and unilaterally in the para-aortic region (n=1). In all four cases, nodal metastases were located within SLNs detected by lymphoscintigraphy. Only 10 of the 26 patients (38%) had significant blue dye staining. All blue-stained SLNs were radioactive. In patients with endometrial cancer, it is feasible to use lymphatic mapping and SLN biopsy to define the topographic distribution of the lymphatic network and also to accurately detect lumbo-aortic and pelvic metastases within SLNs. In the majority of patients with early stage endometrial cancer, this procedure may avoid unnecessary radical pelvic lymphadenectomy. It may also guide para-aortic lymph node dissection on the basis of the SLN status.

Comparison of two methods used to prepare smears of mouse lung tissue for detection of Pneumocystis carinii.

PubMed Central

Thomson, R B; Smith, T F; Wilson, W R

1982-01-01

The laboratory diagnosis of Pneumocystis carinii pneumonia in humans includes the identification of cysts in stained lung tissue impression smears. By using a mouse model, we compared the number of cysts in lung tissue impression smears with those contained in a concentrate of homogenized lung tissue. Eleven C3H/HEN mice developed P. carinii infection after corticosteroid injections, a low protein (8%) diet, and tetracycline administered in drinking water. Impression smears were prepared with freshly bisected lung tissue. Smears of concentrates were prepared with sediment from centrifuged lung tissue homogenates. All smears were made in duplicate, stained with toluidine blue O or methenamine silver, coded, randomized, and examined. The concentrate preparations contained more cysts per microscopic field than the impression preparations (P less than 0.01). Concentrates prepared by grinding with a mortar and pestle contained more cysts than concentrates prepared by blending with a Stomacher (P less than 0.05). Cysts were detected equally well with either the toluidine blue O or silver stain (not significant). Lung tissue concentrates were superior to lung tissue impressions for detecting P. carinii cysts in mice. Use of lung tissue concentrates should be considered for the diagnosis of human P. carinii infection. PMID:6181088

[Pneumocystis pneumonia biological diagnosis at Fann Teaching Hospital in Dakar, Senegal].

PubMed

Dieng, Y; Dieng, T; Sow, D; Wlouhou, S; Sylla, K; Tine, R; Ndiaye, M; Ndiaye, J L; Faye, B; Faye, O; Gaye, O

2016-03-01

Data relative to Pneumocystis pneumonia in sub-Saharan Africa are not well known. Weakness of the technical material and use of little sensitive biological tools of diagnosis are among the evoked reasons. The objective of this study is to update the data of the disease at the Fann Teaching Hospital in Dakar and to estimate biological methods used in diagnosis. A descriptive longitudinal study was carried out from January 5th, 2009 to October 31st, 2011 in the parasitology and mycology laboratory of the Fann Teaching Hospital in Dakar. The bronchoalveolar lavages received in the laboratory were examined microscopically for Pneumocystis jirovecii by indirect fluorescent assay or after Giemsa or toluidine blue O staining. One hundred and eighty-three bronchoalveolar lavages withdrawn from 183 patients were received in the laboratory. Sixteen were positive for P. jirovecii at 9% frequency. Four among these patients were HIV positive. Indirect fluorescent assay allowed finding of P. jirovecii among 16 patients while Giemsa staining discovered P. jirovecii only in a single patient. No case was diagnosed by toluidine blue O staining. Pneumocystis pneumonia in Parasitology and Mycology Laboratory of Fann Teaching Hospital at Dakar was mainly diagnosed among HIV patients. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Diurnal lighting patterns and habitat alter opsin expression and colour preferences in a killifish

PubMed Central

Johnson, Ashley M.; Stanis, Shannon; Fuller, Rebecca C.

2013-01-01

Spatial variation in lighting environments frequently leads to population variation in colour patterns, colour preferences and visual systems. Yet lighting conditions also vary diurnally, and many aspects of visual systems and behaviour vary over this time scale. Here, we use the bluefin killifish (Lucania goodei) to compare how diurnal variation and habitat variation (clear versus tannin-stained water) affect opsin expression and the preference to peck at different-coloured objects. Opsin expression was generally lowest at midnight and dawn, and highest at midday and dusk, and this diurnal variation was many times greater than variation between habitats. Pecking preference was affected by both diurnal and habitat variation but did not correlate with opsin expression. Rather, pecking preference matched lighting conditions, with higher preferences for blue at noon and for red at dawn/dusk, when these wavelengths are comparatively scarce. Similarly, blue pecking preference was higher in tannin-stained water where blue wavelengths are reduced. In conclusion, L. goodei exhibits strong diurnal cycles of opsin expression, but these are not tightly correlated with light intensity or colour. Temporally variable pecking preferences probably result from lighting environment rather than from opsin production. These results may have implications for the colour pattern diversity observed in these fish. PMID:23698009

A novel histological technique for distinguishing between epithelial cells in forensic casework.

PubMed

French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

2008-06-10

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Tissue sulfomucin and sialomucin content in colon mucosa without intestinal transit subjected to intervention with Curcuma longa (curcumin).

PubMed

Alves, Antonio José Tiburcio; Pereira, José Aires; Pansani, Adrieli Heloísa Campardo; Magro, Daniela Oliveira; Coy, Cláudio Saddy Rodrigues; Martinez, Carlos Augusto Real

2017-03-01

To measure the tissue sulfomucin and sialomucin content of the colon mucosa without fecal flow, subjected to intervention with curcumin, and the influence of the concentration used and the intervention time. Thirty-six rats were subjected to proximal right colostomy and distal mucous fistula. They were divided into two groups according to whether sacrifice was performed two or four weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone; curcumin at 50 mg/kg/day or curcumin at 200 mg/kg/day. Acid mucins were diagnosed using the Alcian blue technique. The mucin content was quantified by means of computer-assisted image analysis. The significance level of 5% was used throughout (p < 0.05). There were dose-related increases in the quantities of sulfomucins in the animals subjected to interventions with curcumin, both after two weeks (p < 0.00001) and after four weeks (p < 0.00001). There were increases in sialomucin quantity that were concentration-related (p < 0.00001) and time-related (p < 0.00001). Curcumin enemas increase the quantity of acid mucins in the intestinal flow in the excluded colon, with dose and time dependency.

Is blue dye still required during sentinel lymph node biopsy for breast cancer?

PubMed

Peek, Mirjam Cl; Kovacs, Tibor; Baker, Rose; Hamed, Hisham; Kothari, Ash; Douek, Michael

2016-01-01

In early breast cancer, the optimal technique for sentinel lymph node biopsy (SLNB) is the combined technique (radioisotope and Patent Blue V) which achieves high identification rates. Despite this, many centres have decided to stop using blue dye due to blue-dye-related complications (tattoo, anaphylaxis). We evaluated the SLNB identification rate using the combined technique with and without Patent Blue V and the blue-dye-related complication rates. Clinical and histological data were analysed on patients undergoing SLNB between March 2014 and April 2015. SLNB was performed following standard hospital protocols using the combined technique. A total of 208 patients underwent SLNB and 160 patients (342 nodes) with complete operation notes were available for final analysis. The identification rate with the combined technique was 98.8% ( n = 158/160), with blue dye alone 92.5% ( n = 148/160) and with radioisotope alone 97.5% ( n = 156/160). A total of 76.9% (263/342) of nodes were radioactive and blue, 15.5% (53/342) only radioactive and 2.3% (8/342) only blue, 5.3% (18/342) were neither radioactive nor blue. No anaphylactic reactions were reported and blue skin staining was reported in six (3.8%) patients. The combined technique should continue be the preferred technique for SLNB and should be standardised. Radioisotope alone (but not blue dye alone) has comparable sentinel node identification rates in experienced hands. National guidelines are required to optimise operative documentation.

Epigenetic Mechanisms of Folate Nutrition in Breast Cancer

DTIC Science & Technology

2012-04-01

made we will study the effects of depletion of protein expression on breast cancer cell growth, apoptosis , replication, migration, ability to...AHCY or DNMT. Measured Endpoint Assay Cell proliferation Trypan blue exclusion assay and MTT assay Cell apoptosis TUNEL assay and caspase 3 staining

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wheeler, T.M.; Johnson, E.H.; Coughlin, D.

While asbestos bodies (ABs) in sputum and/or bronchial washings are highly specific markers for significant asbestos exposure, comparison of the sensitivity between sputum cytology and bronchial washing cytology for the detection of ABs had not been documented. Review of the files of the Cytopathology Laboratory, Methodist Hospital, Houston, Texas, for the period 1973 to 1984 identified 11 patients with slides available for review who (1) had been examined by both sputum cytology and bronchial washing cytology and (2) had at least one specimen positive for ABs. Of the 11 evaluable cases, all had ABs in the bronchial washings but onymore » 6 had ABs in the sputum. In addition, iron stain (e.g., the Prussian blue stain) was found to be more sensitive than the Papanicolaou stain for the detection of ABs in these cases. These findings indicate that iron-stained bronchial washing specimens should be preferred for the cytologic detection of asbestos exposure.« less

Structure and Activity Changes of Phytohemagglutinin from Red Kidney Bean (Phaseolus vulgaris) Affected by Ultrahigh-Pressure Treatments.

PubMed

Lu, Yunjun; Liu, Cencen; Zhao, Mouming; Cui, Chun; Ren, Jiaoyan

2015-11-04

Phytohemagglutin (PHA), purified from red kidney beans (Phaseolus vulgaris) by Affi-Gel blue affinity chromatography, was subjected to ultrahigh-pressure (UHP) treatment (150, 250, 350, and 450 MPa). The purified PHA lost its hemagglutination activity after 450 MPa treatment and showed less pressure tolerance than crude PHA. However, the saccharide specificity and α-glucosidase inhibition activity of the purified PHA did not change much after UHP treatment. Electrophoresis staining by periodic acid-Schiff (PAS) manifested that the glycone structure of purified PHA remained stable even after 450 MPa pressure treatment. However, electrophoresis staining by Coomassie Blue as well as circular dichroism (CD) and differential scanning calorimetry (DSC) assay proved that the protein unit structure of purified PHA unfolded when treated at 0-250 MPa but reaggregates at 250-450 MPa. Therefore, the hemagglutination activity tends to be affected by the protein unit structure, while the stability of the glycone structure contributed to the remaining α-glucosidase inhibition activity.

CCD Camera Detection of HIV Infection.

PubMed

Day, John R

2017-01-01

Rapid and precise quantification of the infectivity of HIV is important for molecular virologic studies, as well as for measuring the activities of antiviral drugs and neutralizing antibodies. An indicator cell line, a CCD camera, and image-analysis software are used to quantify HIV infectivity. The cells of the P4R5 line, which express the receptors for HIV infection as well as β-galactosidase under the control of the HIV-1 long terminal repeat, are infected with HIV and then incubated 2 days later with X-gal to stain the infected cells blue. Digital images of monolayers of the infected cells are captured using a high resolution CCD video camera and a macro video zoom lens. A software program is developed to process the images and to count the blue-stained foci of infection. The described method allows for the rapid quantification of the infected cells over a wide range of viral inocula with reproducibility, accuracy and at relatively low cost.

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Characterization of Uterine Granular Cell Tumors in B6C3F1 Mice: A Histomorphologic, Immunohistochemical, and Ultrastructural Study

PubMed Central

Veit, A. C.; Painter, J. T.; Miller, R. A.; Hardisty, J. F.; Dixon, D.

2009-01-01

The granular cell tumor is most often a benign neoplasm of uncertain origin. Four uterine granular cell tumors in control and treated female B6C3F1 mice were identified in chronic studies at the National Toxicology Program. Two tumors occurred in untreated control animals and 2 in treated animals receiving different compounds. Tissue sections were evaluated histologically and stained with hematoxylin and eosin, periodic acid–Schiff with diastase resistance, Masson’s trichrome, toluidine blue, phosphotungstic acid–hematoxylin, and stained immunohistochemically with a panel of antibodies to muscle (desmin, alpha smooth muscle actin), neural (S-100, neuron specific enolase), epithelial (wide-spectrum cytokeratin), and macrophage (F4/80) markers. The main histomorphologic feature of tumor cells was the presence of abundant cytoplasmic eosinophilic granules that stained positive for periodic acid–Schiff with diastase resistance. Tumors varied in appearance and were comprised of sheets and nests of round to polygonal cells with distinct borders. Nuclei were hyperchromatic, pleomorphic, and centrally to eccentrically located and often contained single nucleoli. Occasional multinucleated giant cells were observed. Tumors were pale pink and homogeneous with trichrome stain and negative with toluidine blue. Three tumors had positive to weakly positive immunoreactivity for desmin, and 1 was positive for alpha smooth muscle actin. Expression of S-100, wide-spectrum cytokeratin, and neuron-specific enolase was negative for all tumors. Ultrastructurally, prominent electron-dense cytoplasmic granules were abundant and contained secondary lysosomes with heterogeneous lysosomal contents. The characteristics of these uterine granular cell tumors were suggestive of a myogenic origin. PMID:18725470

PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

EPA Science Inventory

Abstract

Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

Visual and anatomical outcome of macular hole surgery at a tertiary healthcare facility.

PubMed

Kumari, Komalta; Tahir, Muhammad Ali; Cheema, Alyscia

2017-01-01

To assess visual and anatomical outcome of full thickness macular hole (FTMH) surgery with ILM peeling using brilliant blue G dye. Thirty patients who had clinically evident macular hole were selected. Pre-operative Optical Coherence Tomography (OCT) was done. In all cases vitrectomy was performed via 23guage 3 ports pars plana (3PPV) vitrectomy system and Brilliant blue G dye, 0.5ml dye was injected over macula which resulted in light blue stain of ILM and peeling was performed around hole in circular motion and after gas fluid exchange gas tamponade with SF6 was done. Final visual and anatomical outcome was measured as postoperative BCVA and postoperative OCT at three months respectively. Descriptive statistics were computed. Paired t-test was applied. P value≤0.05 were considered as significant. There were 12 male and 18 female patients. The mean age was 57.40±4.76 years. The mean size of macular hole was 452.20±242.33μm. The mean duration of symptoms was 16.73±13.49 weeks. Mean pre operative BCVA was 1.30±0.73 log MAR and post operative was 0.51±0.23 log MAR. Mean increased BCVA was found to be 0.22±0.13 log MAR. Primary closure of hole was achieved in 29(96.7%). Significant mean difference was found in pre operative and post operative BCVA. Brilliant blue G exhibits sufficient staining qualities and safety profile to peel ILM in the management of full thickness macular hole with significant visual and anatomical improvement.

Light microscopy with differential staining techniques for the characterisation and discrimination of insects versus marine arthropods processed animal proteins.

PubMed

Ottoboni, Matteo; Tretola, Marco; Cheli, Federica; Marchis, Daniela; Veys, Pascal; Baeten, Vincent; Pinotti, Luciano

2017-08-01

The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods - classified as fishmeal. Specifically, three samples of single-species insect material, Hermetia illucens (HI), Bombyx mori (BM) and Tenebrio molitor (TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed.

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

PubMed

Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

2016-01-01

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

A Simplified Experimental Scheme for the Study of Mitosis.

ERIC Educational Resources Information Center

Gill, John

1980-01-01

A procedure is described for providing preparations of dividing cells from root apical meristems, requiring only inexpensive equipment and minimal experimental skill, and using 8-Hydroxyquinoline and Toluidene-blue as a chromosome stain. The method has been sucessfully tested in schools and yields permanent preparations of adequate quality for…

Identification of oleoresin in epoxy-embedded slash pine tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birchem, R.; Brown, C.L.

1978-01-01

Sudan black B stains oleoresin blue-black in epoxy-embedded material as well as in living tissue. The Sudan black B staining properties of oleoresin are similar to those of lipid, but it can be distinguished from tannin, which stains brown. Practically all oleoresin present in resin ducts and intercellular spaces, and much of that contained in epithelial and ray cells, is extracted in preparatory procedures for electron microscopy. A fixation procedure is proposed which preserves significantly more oleoresin in situ. The use of Sudan black B enables one to localize oleoresin by light microscopy, and permits direct comparison of adjacent sectionsmore » of epoxy-embedded material at the ultrastructure level. Ultrastructurally oleoresin and lipid possess similar electron densities and can be distinguished from the highly electron-opaque tannin deposits.« less

A case of epidermal cyst with pilomatrical differentiation.

PubMed

Ikoma, Norihiro; Iwashita, Kenichi; Umezawa, Yoshinori; Matsuyama, Takashi; Ohta, Yukinori; Ozawa, Akira; Umemura, Shinobu; Ueyama, Yoshito; Yamazaki, Hitoshi

2004-09-01

A 20-year-old Japanese woman with an epidermal cyst on the back is described. Physical examination revealed a deep blue and round shaped cystic lesion measuring 10 min in diameter. A comedo-like keratotic plug also could be seen at the center. Histologically, the inner surface of the cyst was clearly separated of two types of the cells. The one was layers of epidermal keratinocytes and the other looked like a basal layer of epidermis, which immunohistochemically stained by S-100, HMB-45, cytokeratin (CK19) and Fontana-Masson staining. We diagnosed this case as epidermal cyst with pilomatrical differentiation.

Influence of orthodontic adhesives and clean-up procedures on the stain susceptibility of enamel after debonding.

PubMed

Joo, Hyun-Jin; Lee, Yong-Keun; Lee, Dong-Yul; Kim, Yae-Jin; Lim, Yong-Kyu

2011-03-01

To determine the influence of the type of orthodontic adhesive system, such as conventional acid-etching (CE) and self-etching primers (SEPs), on the stain susceptibility of enamel surface after debonding. Effects of clean-up procedures on the enamel surface were also determined. Two types and four brands of adhesive systems were investigated using 135 human premolars. Unbonded teeth were used as controls. Three-dimensional scanning of the enamel surface was performed before bracket bonding, after debonding, and after clean-up procedures. The color of each tooth was measured before bracket bonding and again after debonding and clean-up procedures. This was followed by methylene blue staining. The stain susceptibility of the enamel surface was measured after finishing only (F-condition) and after finishing/polishing (FP-condition). After debonding, the amount of residual adhesive resins in CE materials was greater than that in SEP materials. For the F-condition, staining color change in SEP materials was significantly higher than that in CE materials. For the FP-condition, staining color change in both CE and SEP materials was not different from those of the control. The SEP system would show less stain susceptibility if the thin residual adhesive resin layer after debonding is removed by polishing.

Inhibition of iron overload-induced apoptosis and necrosis of bone marrow mesenchymal stem cells by melatonin.

PubMed

Yang, Fan; Li, Yuan; Yan, Gege; Liu, Tianyi; Feng, Chao; Gong, Rui; Yuan, Ye; Ding, Fengzhi; Zhang, Lai; Idiiatullina, Elina; Pavlov, Valentin; Han, Zhenbo; Ma, Wenya; Huang, Qi; Yu, Ying; Bao, Zhengyi; Wang, Xiuxiu; Hua, Bingjie; Du, Zhimin; Cai, Benzhi; Yang, Lei

2017-05-09

Iron overload induces severe damage to several vital organs such as the liver, heart and bone, and thus contributes to the dysfunction of these organs. The aim of this study is to investigate whether iron overload causes the apoptosis and necrosis of bone marrow mesenchymal stem cells (BMSCs) and melatonin may prevent its toxicity. Perls' Prussion blue staining showed that exposure to increased concentrations of ferric ammonium citrate (FAC) induced a gradual increase of intracellular iron level in BMSCs. Trypan blue staining demonstrated that FAC decreased the viability of BMSCs in a concentration-dependent manner. Notably, melatonin protected BMSCs against apoptosis and necrosis induced by FAC and it was vertified by Live/Dead, TUNEL and PI/Hoechst stainings. Furthermore, melatonin pretreatment suppressed FAC-induced reactive oxygen species accumulation. Western blot showed that exposure to FAC resulted in the decrease of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bax and Cleaved Caspase-3, and necrosis-related proteins RIP1 and RIP3, which were significantly inhibited by melatonin treatment. At last, melatonin receptor blocker luzindole failed to block the protection of BMSCs apoptosis and necrosis by melatonin. Taken together, melatonin protected BMSCs from iron overload induced apoptosis and necrosis by regulating Bcl-2, Bax, Cleaved Caspase-3, RIP1 and RIP3 pathways.

Inhibition of iron overload-induced apoptosis and necrosis of bone marrow mesenchymal stem cells by melatonin

PubMed Central

Yan, Gege; Liu, Tianyi; Feng, Chao; Gong, Rui; Yuan, Ye; Ding, Fengzhi; Zhang, Lai; Idiiatullina, Elina; Pavlov, Valentin; Han, Zhenbo; Ma, Wenya; Huang, Qi; Yu, Ying; Bao, Zhengyi; Wang, Xiuxiu; Hua, Bingjie; Du, Zhimin; Cai, Benzhi; Yang, Lei

2017-01-01

Iron overload induces severe damage to several vital organs such as the liver, heart and bone, and thus contributes to the dysfunction of these organs. The aim of this study is to investigate whether iron overload causes the apoptosis and necrosis of bone marrow mesenchymal stem cells (BMSCs) and melatonin may prevent its toxicity. Perls’ Prussion blue staining showed that exposure to increased concentrations of ferric ammonium citrate (FAC) induced a gradual increase of intracellular iron level in BMSCs. Trypan blue staining demonstrated that FAC decreased the viability of BMSCs in a concentration-dependent manner. Notably, melatonin protected BMSCs against apoptosis and necrosis induced by FAC and it was vertified by Live/Dead, TUNEL and PI/Hoechst stainings. Furthermore, melatonin pretreatment suppressed FAC-induced reactive oxygen species accumulation. Western blot showed that exposure to FAC resulted in the decrease of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bax and Cleaved Caspase-3, and necrosis-related proteins RIP1 and RIP3, which were significantly inhibited by melatonin treatment. At last, melatonin receptor blocker luzindole failed to block the protection of BMSCs apoptosis and necrosis by melatonin. Taken together, melatonin protected BMSCs from iron overload induced apoptosis and necrosis by regulating Bcl-2, Bax, Cleaved Caspase-3, RIP1 and RIP3 pathways. PMID:28415572

Questions on unusual Mimivirus-like structures observed in human cells.

PubMed

Lusi, Elena Angela; Maloney, Dan; Caicci, Federico; Guarascio, Paolo

2017-01-01

Background: Mimiviruses or giant viruses that infect amoebas have the ability to retain the Gram stain, which is usually used to colour bacteria. There is some evidence suggesting that Mimiviruses can also infect human cells. Guided by these premises, we performed a routine Gram stain on a variety of human specimens to see if we could detect the same Gram positive blue granules that identify Mimiviruses in the amoebas.  Methods: We analysed 24 different human specimens (liver, brain, kidney, lymph node and ovary) using Gram stain histochemistry, electron microscopy immunogold, high resolution mass spectrometry and protein identification.  Results: We detected in the human cells Gram positive granules that were distinct from bacteria. The fine blue granules displayed the same pattern of the Gram positive granules that diagnose Mimiviruses in the cytoplasm of the amoebas. Electron microscopy confirmed the presence of human Mimiviruses-like structures and mass spectrometry identified histone H4 peptides, which had the same footprints as giant viruses. However, some differences were noted: the Mimivirus-like structures identified in the human cells were ubiquitous and manifested a distinct mammalian retroviral antigenicity.  Conclusions: Our main hypotheses are that the structures could be either giant viruses having a retroviral antigenicity or ancestral cellular components having a viral origin. However, other possible alternatives have been proposed to explain the nature and function of the newly identified structures.

[Osteological development of the vertebral column and caudal complex of Lujanus guttatus (Perciformes: Lutjanidae) larvae under rearing conditions].

PubMed

Rodríguez-Ibarra, Luz Estela; Abdo-de la Parra, María Isabel; Aguilar-Zárate, Gabriela; Valasco-Blanco, Gabriela; Ibarra-Castro, Leonardo

2015-03-01

The spotted rose snapper (Lutjanus guttatus) is an important commercial species in Mexico with good culture potential. The osteological study at early stages in this species is an important tool to confirm normal bone structure and for the detection of malformations that may occur during early development. This study was carried out in order to evaluate and describe the normal osteological development of the vertebral column and caudal complex of this species grown under controlled conditions. For this, a total of 540 larvae of L. guttatus, between 2.1 and 17.5 mm of total length (TL), were cultured during 36 days; culture conditions were 28 degrees C, 5.74 mg/L oxygen and 32.2 ups salinity with standard feeding rates. To detect growth changes, a sample of 15 organisms was daily taken from day one until day 36 of post-hatch (DPH). Samples were processed following standard techniques of clearing, and cartilage (alcian blue) and bone staining (alizarin red). Results showed that the vertebral column is composed of ten vertebrae in the abdominal region, and 14 vertebrae including the urostyle in the caudal region. The development of the axial skeleton starts with the neural arches and haemal arches at 3.8 mm TL. Caudal elements such as the hypurals and parahypural began to develop at 4.1 mm TL. Pre-flexion and flexion of the notochord and the formation of all hypurals were observed between 5.3 and 5.8 mm TL. Ossification of the vertebrae in the abdominal region and in some neural arches initiated at 9.5mm TL. In the caudal region, all the neural and haemal arches ossified at 10.2 mm TL. All the abdominal vertebrae and their respective neural arches and parapophyses ossified at 11.2 mm TL, while the elements of the caudal complex that ossified were the hypurals, parahypurals and modified haemal spines. All caudal fm rays, 12 neural spines and 3 haemal arches were ossified by 15.5 mm. The complete ossification process of this specie under laboratory culture conditions was observed when larvae reached 17.3 mm TL on 36 DPH. Detailed analysis of the osteological structures will allow a reference description to evaluate and detect malformations that may occur during the larval culture of the spotted rose snapper.

Three-dimensional structure of human lamellar bone: the presence of two different materials and new insights into the hierarchical organization.

PubMed

Reznikov, Natalie; Shahar, Ron; Weiner, Steve

2014-02-01

Lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the long axis of the bone, and aligned within 10-20° of the long axis, as well as fanning arrays. At a higher organizational level, arrays of ordered collagen fibrils are organized into 'rods' around 2 to 3μm in diameter, and the long axes of these 'rods' are parallel to the lamellar boundaries. Human bone also contains a disordered component that envelopes the rods and fills in the spaces between them. The disordered motif is especially well-defined between adjacent layers of rods. The disordered motif and its interfibrillar substance stain heavily with osmium tetroxide and Alcian blue indicating the presence of another organic component in addition to collagen. The canalicular network is confined to the disordered material, along with voids and individual collagen fibrils, some of which are also aligned more or less perpendicular to the lamellar boundaries. The organization of the ordered fibril arrays into rods enveloped in the continuous disordered structure was not observed in rat lamellar bone. We thus conclude that human lamellar bone is comprised of two distinct materials, an ordered material and a disordered material, and contains an additional hierarchical level of organization composed of arrays of ordered collagen fibrils, referred to as rods. This new structural information on human lamellar bone will improve our understanding of structure-mechanical function relations, mechanisms of mechano-sensing and the characterizations of bone pathologies. Copyright © 2013 Elsevier Inc. All rights reserved.

Methylene Blue: The Long and Winding Road From Stain to Brain: Part 2.

PubMed

Howland, Robert H

2016-10-01

Methylene blue was the first synthetic drug ever used in medicine, having been used to treat clinical pain syndromes, malaria, and psychotic disorders more than one century ago. Methylene blue is a cationic thiazine dye with redox-cycling properties and a selective affinity for the nervous system. This drug also inhibits the activity of monoamine oxidase, nitric oxide synthase, and guanylyl cyclase, as well as tau protein aggregation; increases the release of neurotransmitters, such as serotonin and norepinephrine; reduces amyloid-beta levels; and increases cholinergic transmission. The action of methylene blue on multiple cellular and molecular targets justifies its investigation in various neuropsychiatric disorders. Investigations of methylene blue were instrumental in the serendipitous development of phenothiazine antipsychotic drugs. Although chlorpromazine is heralded as the first antipsychotic drug used in psychiatry, methylene blue is a phenothiazine drug that had been used to treat psychotic patients half a century earlier. It has also been studied in bipolar disorder and deserves further investigation for the treatment of unipolar and bipolar disorders. More recently, methylene blue has been the subject of preclinical and clinical investigations for cognitive dysfunction, dementia, and other neurodegenerative disorders. [Journal of Psychosocial Nursing and Mental Health Services, 54(10), 21-26.]. Copyright 2016, SLACK Incorporated.

Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation; mass spectrometry analysis of oxidation products.

PubMed

Tacal, Ozden; Li, Bin; Lockridge, Oksana; Schopfer, Lawrence M

2013-01-01

Methylene blue, 3, 7-bis(dimethylamino)-phenothiazin-5-ium chloride, is a reversible inhibitor of human butyrylcholinesterase (BChE) in the absence of light. In the presence of light and oxygen, methylene blue promotes irreversible inhibition of human BChE as a function of time, requiring 3 h irradiation to inhibit 95% activity. Inactivation was accompanied by a progressive loss of Coomassie-stained protein bands on native and denaturing polyacrylamide gels, suggesting backbone fragmentation. Aggregation was not detected. MALDI-TOF/TOF mass spectrometry identified oxidized tryptophan (W52, 56, 231, 376, 412, 490, 522), oxidized methionine (M81, 144, 302, 532, 554, 555), oxidized histidine (H214), oxidized proline (P230), oxidized cysteine (C519) and oxidized serine (S215). A 20 min irradiation in the presence of methylene blue resulted in 17% loss of BChE activity, suggesting that BChE is relatively resistant to methylene blue-catalyzed photoinactivation and that therefore this process could be used to sterilize BChE preparations. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

PubMed

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study.

PubMed

Desoubeaux, Guillaume; Franck-Martel, Claire; Caille, Agnès; Drillaud, Nicolas; Lestrade Carluer de Kyvon, Marie-Alix; Bailly, Éric; Chandenier, Jacques

2017-04-01

The biological diagnosis of Pneumocystis jirovecii pneumonia (PjP) is based on the investigation of respiratory fluids by conventional staining methods and/or molecular biology. Diagnostic performance of an in-house technique based on calcofluor-blue brightener for the direct detection of P. jirovecii cysts was prospectively assessed in bronchial-alveolar lavage fluids (BALF) from patients with a suspected PjP infection over a three-year period in a single center: the diagnostic yield was compared to that of a commercial kit based on monoclonal immunofluorescence assay (IFA) on replicate smears. May-Grünwald Giemsa (MGG) staining and quantitative Polymerase Chain Reaction (qPCR) were also performed. The gold standard for each patient was the definitive diagnosis of PjP infection by an independent committee based on clinical, radiological, and biological data. Overall, 481 BALF were assessed: 42 were found to be positive for the detection of P. jirovecii by at least one laboratory technique, but only 35 were actually judged to be in agreement with the definitive diagnosis of PjP infection. The sensitivity of the calcofluor-blue brightener technique was 74.3% vs. 60.0%, 34.6%, and 82.9% for IFA, MGG, and qPCR, respectively; and its specificity was 99.6% vs. 99.3%, 100.0%, and 99.4% for IFA, MGG, and qPCR. No technique was shown to be statistically superior to calcofluor-blue brightener. Further validation of the test through multicenter studies is now required, but in light of its low cost and easy preparation, the use of calcofluor-blue brightener in BALF appears to be a valuable alternative method for the routine first-line diagnosis of PjP infection. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effect of ultrasound sonication on clonogenic survival and mitochondria of ovarian cancer cells in the presence of methylene blue.

PubMed

Xiang, Junyan; Leung, Albert Wingnang; Xu, Chuanshan

2014-10-01

This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P < .05). Ultrasound sonication in the presence of methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells. © 2014 by the American Institute of Ultrasound in Medicine.

[Not Available].

PubMed

Charbonneau, Annie

2013-07-01

Methylene blue is used in medical practice for various reasons. Recent findings point to a potential interaction with serotonin reuptake inhibitors (SRIs) that could lead to serotonergic toxicity. To describe the risk of serotonergic toxicity associated with the interaction between methylene blue and SRIs. Relevant publications were searched systematically via MEDLINE (1946 to March 21, 2013) and Embase (1974 to 2013, week 11) with the following search terms: "methylene blue", "methylthioninium", "monoamine oxidase inhibitors", "serotonin reup-take inhibitors", and "serotonin syndrome". No restrictions were applied in relation to the indication for methylene blue or the language of publication. The reference lists of identified articles were also searched. Eighteen case reports and 2 case series were identified for inclusion. To date, no randomized controlled trials have been published. The first case report indicating suspicion of an interaction between methylene blue and SRIs was published in 2003. Seventeen other case reports describing the same type of interaction have been published since then. The 2 case series provided data from about 325 parathyroidectomies in which methylene blue was used for staining. The 17 patients who experienced central nervous system toxicity were all taking SRIs in the preoperative period. When administered in combination with SRIs, methylene blue may lead to serotonergic toxicity at doses as low as 0.7 mg/kg. Methylene blue would seem to have monoamine oxidase A inhibitory properties. Precautions should be taken to avoid this interaction. [Publisher's translation].

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

PubMed

Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

An evaluation of novel vital dyes for intraocular surgery.

PubMed

Haritoglou, Christos; Yu, Alice; Freyer, Wolfgang; Priglinger, Siegfried G; Alge, Claudia; Eibl, Kirsten; May, Christian A; Welge-Luessen, Ulrich; Kampik, Anselm

2005-09-01

To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery. Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (ARPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%. All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro. The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

Sentinel lymph node detection in early stage cervical cancer: a prospective study comparing preoperative lymphoscintigraphy, intraoperative gamma probe, and blue dye.

PubMed

Kara, P Pelin; Ayhan, Ali; Caner, Biray; Gültekin, Murat; Ugur, Omer; Bozkurt, M Fani; Usubutun, Alp

2008-07-01

The objective of this prospective study was to determine the feasibility of sentinel lymph node (SLN) detection in patients with cervical cancer using lymphoscintigraphy (LS), gamma probe, and blue dye. A total of 32 patients with early stage cervical cancer (FIGO IA2-IIA) who were treated with total abdominal hysterectomy and bilateral pelvic and paraortic lymphadenectomy underwent SLN biopsy. LS was performed on all the patients following the injection of 74 MBq technetium-99m-nanocolloid pericervically. The first appearing persistent focal accumulation on either dynamic or static images of LS was considered to be an SLN. Blue dye was injected just prior to surgical incision in 16 patients (50%) at the same locations as the radioactive isotope injection. During the operation, blue-stained node(s) were excised as SLNs. For gamma probe, a lymph node was accepted as an SLN, if its ex vivo radioactive counts were at least 10-fold above background radioactivity. SLNs, which were negative by routine hematoxylin and eosin (H&E) examination, were histopathologically reevaluated for the presence of micrometastases by step sectioning and immunohistochemical staining with pancytokeratin. At least one SLN was identified for each patient by gamma probe. Intraoperative gamma probe was the most sensitive method with a technical success rate of SLN detection of 100% (32/32), followed by LS 87.5% (28/32) and blue dye 68.8% (11/16), respectively. The average number of SLNs per patient detected by gamma probe was 2.09 (range 1-5). The localizations of the SLNs were external iliac 47.8%, obturatory 32.8%, common iliac 9%, paraaortic 4.4%, and paracervical 6%. Micrometastases, not detected by routine H&E were found by immunohistochemistry in one patient. On the basis of the histopathological analysis, the negative predictive value for predicting metastases was 100%, and there were no false-negative results. Preoperative LS with radiocolloids, intraoperative lymphatic mapping with blue dye and gamma probe are all feasible methods comparable with each other for SLN detection in early stage cervical cancer patients, but gamma probe is the most useful method in terms of technical success.

Assessing the use of Quantitative Light-induced Fluorescence-Digital as a clinical plaque assessment.

PubMed

Han, Sun-Young; Kim, Bo-Ra; Ko, Hae-Youn; Kwon, Ho-Keun; Kim, Baek-Il

2016-03-01

The aims of this study were to compare the relationship between red fluorescent plaque (RF plaque) area by Quantitative Light-induced Fluorescence-Digital (QLF-D) and disclosed plaque area by two-tone disclosure, and to assess the bacterial composition of the RF plaque by real time-PCR. Fifty healthy subjects were included and 600 facial surfaces of their anterior teeth were examined. QLF-D was taken on two separate occasions (before and after disclosing), and the RF plaque area was calculated based on Plaque Percent Index (PPI). After disclosing, the stained plaque area was analyzed to investigate the relationship with the RF plaque area. The relationship was evaluated using Pearson correlation and paired t-test. Then, the RF and non-red fluorescent (non-RF) plaque samples were obtained from the same subject for real-time PCR test. Total 10 plaque samples were compared the ratio of the 6 of bacteria using Wilcoxon signed rank test. Regarding the paired t-test, the blue-staining plaque area (9.3±9.2) showed significantly similarity with the RF plaque area (9.1±14.9, p=0.80) at ΔR20, however, the red-staining plaque area (31.6±20.9) presented difference from the RF plaque area (p<0.0001). In addition, bacterial composition of Prevotella intermedia and Streptococcus anginosus was associated with substantially more the RF plaque than the non-RF plaque (p<0.05). The plaque assessment method using QLF-D has potential to detect mature plaque, and the plaque area was associated with the blue-staining area using two-tone disclosure. Copyright © 2015 Elsevier B.V. All rights reserved.

Spectral imaging perspective on cytomics.

PubMed

Levenson, Richard M

2006-07-01

Cytomics involves the analysis of cellular morphology and molecular phenotypes, with reference to tissue architecture and to additional metadata. To this end, a variety of imaging and nonimaging technologies need to be integrated. Spectral imaging is proposed as a tool that can simplify and enrich the extraction of morphological and molecular information. Simple-to-use instrumentation is available that mounts on standard microscopes and can generate spectral image datasets with excellent spatial and spectral resolution; these can be exploited by sophisticated analysis tools. This report focuses on brightfield microscopy-based approaches. Cytological and histological samples were stained using nonspecific standard stains (Giemsa; hematoxylin and eosin (H&E)) or immunohistochemical (IHC) techniques employing three chromogens plus a hematoxylin counterstain. The samples were imaged using the Nuance system, a commercially available, liquid-crystal tunable-filter-based multispectral imaging platform. The resulting data sets were analyzed using spectral unmixing algorithms and/or learn-by-example classification tools. Spectral unmixing of Giemsa-stained guinea-pig blood films readily classified the major blood elements. Machine-learning classifiers were also successful at the same task, as well in distinguishing normal from malignant regions in a colon-cancer example, and in delineating regions of inflammation in an H&E-stained kidney sample. In an example of a multiplexed ICH sample, brown, red, and blue chromogens were isolated into separate images without crosstalk or interference from the (also blue) hematoxylin counterstain. Cytomics requires both accurate architectural segmentation as well as multiplexed molecular imaging to associate molecular phenotypes with relevant cellular and tissue compartments. Multispectral imaging can assist in both these tasks, and conveys new utility to brightfield-based microscopy approaches. Copyright 2006 International Society for Analytical Cytology.

Casparian bands occur in the periderm of Pelargonium hortorum stem and root.

PubMed

Meyer, Chris J; Peterson, Carol A

2011-04-01

Casparian bands are characteristic of the endodermis and exodermis of roots, but also occur infrequently in other plant organs, for example stems and leaves. To date, these structures have not been detected in phellem cells of a periderm. The aim of this study was to determine whether Casparian bands occur in phellem cells using tests that are known to detect Casparian bands in cells that also contain suberin lamellae. Both natural periderm and wound-induced structures were examined in shoots and roots. Using Pelargonium hortorum as a candidate species, the following tests were conducted: (1) staining with berberine and counterstaining with aniline blue, (2) mounting sections in concentrated sulphuric acid and (3) investigating the permeability of the walls with berberine as an apoplastic, fluorescent tracer. (1) Berberine-aniline blue staining revealed a modification in the radial and transverse walls of mature phellem cells in both stems and roots. Three days after wounding through to the cortex of stems, the boundary zone cells (pre-existing, living cells nearest the wound) had developed vividly stained primary walls. By 17 d, staining of mature phellem cells of wound-induced periderm was similar to that of natural periderm. (2) Mature native phellem cells of stems resisted acid digestion. (3) Berberine was excluded from the anticlinal (radial and transverse) walls of mature phellem cells in stems and roots, and from the wound-induced boundary zone. Casparian bands are present in mature phellem cells in both stems and roots of P. hortorum. It is proposed that Casparian bands act to retard water loss and pathogen entry through the primary cell walls of the phellem cells, thus contributing to the main functions of the periderm.

A staining protocol for identifying secondary compounds in Myrtaceae1

PubMed Central

Retamales, Hernan A.; Scharaschkin, Tanya

2014-01-01

• Premise of the study: Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Methods and Results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. • Conclusions: Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements. PMID:25309840

Bark beetle-induced forest mortality in the North American Rocky Mountains

Treesearch

Kevin Hyde; Scott Peckham; Tom Holmes; Brent Ewers

2016-01-01

The epidemic of mortality by insects and disease throughout the Northern American Rocky Mountains exceeds previous records both in severity and spatial extent. Beetle attacks weaken trees and introduce blue-stain fungi that induce hydraulic failure leading to mortality. The magnitude of this outbreak spurs predictions of major changes to...

Fluorescent Magnesium Nanocomplex in Protein Scaffold for Cell Nuclei Imaging Application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandya, Alok; Tripathi, Apritam; Purohit, Rahul

2015-10-27

Here in, we report a facile strategy for the synthesis of water-soluble ultra-fine blue emitting fluorescent Magnesium nanoparticles-protein complex (MgNC). This MgNC is demonstrated to exhibit excellent photo stability and biocompatibility. It was also observed that MgNC stain cell nuclei with high specifcity.

MRI-tracking of transplanted human ASC in a SCID mouse model

NASA Astrophysics Data System (ADS)

Siegmund, Birte J.; Kasten, Annika; Kühn, Jens-Peter; Winter, Karsten; Grüttner, Cordula; Frerich, Bernhard

2017-04-01

Additional Prussian blue stain showed iron in all implants. Significant differences between the three groups (significance level p<0.017) were found after twelve days between control group and group 3 (p=0.008) and after 28 days between control group and group 2 and 3 (p=0.011).

Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

NASA Astrophysics Data System (ADS)

Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

2006-02-01

Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

Effects of cilostamide and/or forskolin on the meiotic resumption and development competence of growing ovine oocytes selected by brilliant cresyl blue staining.

PubMed

Azari-Dolatabad, Nima; Rahmani, H R; Hajian, M; Ostadhosseini, S; Hosseini, S M; Nasr-Esfahani, M H

2016-05-01

The relevance of low developmental competence of in vitro-matured oocyte to the incomplete/delayed cytoplasmic maturation, and the heterogeneity of retrieved oocytes is well established in several species. A short phase of prematuration culture was used to allow better oocyte cytoplasmic maturation. The preselection of growing and fully grown oocytes has been proposed to improve developmental competency. This study investigated the effects of phosphodiesterase type 3-specific inhibitor, cilostamide, and adenylate cyclase activator, forskolin, on the resumption of meiosis and developmental competence of growing ovine oocytes selected by brilliant cresyl blue (BCB) staining. Results indicate that cilostamide, forskolin, and their combination significantly (P < 0.05) increased the percentage of growing (BCB-) oocytes maintained at the germinal vesicle stage. However, only forskolin significantly (P < 0.05) increased the yield and quality of blastocysts derived from BCB- oocytes compared with non-BCB-treated oocytes. We conclude that a short prematuration culture with forskolin may improve the in vitro developmental competency of growing oocytes in ovine. Copyright © 2016 Elsevier Inc. All rights reserved.

Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa.

PubMed

Mahfouz, Reda Z; Sharma, Rakesh K; Said, Tamer M; Erenpreiss, Juris; Agarwal, Ashok

2009-04-01

To examine the relationship among sperm apoptosis, sperm chromatin status, and DNA ploidy in different sperm fractions. Prospective study. Reproductive research center in a tertiary care hospital. Sperm prepared by density gradient were evaluated for sperm count, motility, apoptosis, and sperm chromatin assessment. Sperm count, sperm motility, toluidine blue (TB) results, DNA fragmentation index (%DFI), high DNA stainability, DNA cytometry, and early and late apoptosis. Sperm motility was related to late apoptotic and subhaploid apoptotic sperm (r = -0.56 and -0.53, respectively). The sperm %DFI showed significant correlation with late apoptotic and subhaploid sperm (r = 0.62 and 0.68). TB-stained sperm were significantly correlated with late apoptotic sperm (r = 0.51). Significantly higher proportions of haploid sperm and light blue TB-stained sperm were seen in mature compared with immature fractions. Even in semen samples with low %DFI, semen processing results in a lower incidence of nuclear immaturity and subhaploidy, but the incidence of late apoptotic sperm remains unchanged. Therefore, simultaneous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproductive procedures.

Modified use of methylene blue in the tissue compression technique to detect sarcocysts in meat-producing animals.

PubMed

Ng, Yit Han; Subramaniam, Vellayan; Lau, Yee Ling

2015-11-30

Sarcocystosis in meat-producing animals is a major cause of reduced productivity in many countries, especially those that rely on agriculture. Although several diagnostic methods are available to detect sarcocystosis, many are too time-consuming for routine use in abattoirs and meat inspection centers, where large numbers of samples need to be tested. This study aimed to compare the sensitivity of the methylene blue tissue preparation, unstained tissue preparation and nested PCR in the detection of sarcocysts in tissue samples. Approximately three-fold more sarcocysts were detected in methylene blue-stained tissue compared to unstained controls (McNemar's test: P<0.01). Test sensitivity was comparable to that of the gold standard for sarcocyst detection, nested polymerase chain reaction. These results suggest that methylene blue can be used in tissue compression as a rapid, safe, and inexpensive technique for the detection of ruminant sarcocystosis in abattoirs. Copyright © 2015 Elsevier B.V. All rights reserved.

Sentinel lymph node detection using methylene blue in patients with early stage cervical cancer.

PubMed

Yuan, Song-Hua; Xiong, Ying; Wei, Mei; Yan, Xiao-Jian; Zhang, Hui-Zhong; Zeng, Yi-Xin; Liang, Li-Zhi

2007-07-01

To evaluate the feasibility of sentinel lymph node (SLN) detection in patients with cervical cancer using the low-cost methylene blue dye and to optimize the application procedure. Patients with stage Ib(1)-IIa cervical cancer and subjected to abdominal radical abdominal hysterectomy and pelvic lymphadenectomy were enrolled. Methylene blue, 2-4 ml, was injected into the cervical peritumoral area in 77 cases (4 ml patent blue in the other four cases) 10-360 min before the incision, and surgically removed lymph nodes were examined for the blue lymph nodes that were considered as SLNs. High SLN detection rate was successfully achieved when 4 ml of methylene blue was applied (93.9%, 46/49). Bilaterally SLN detection rate was significantly higher (78.1% vs. 47.1% P=0.027) in cases when the timing of application was more than 60 min before surgery than those with timing no more than 30 min. The blue color of methylene blue-stained SLNs sustained both in vivo and ex vivo, compared with the gradually faded blue color of patent blue that detected in 3 of 4 cases unilaterally. In the total of 112 dissected sides, the most common location of SLNs was the obturator basin (65.2%, 73/112), followed by external iliac area (30.4%, 34/112) and internal iliac area (26.8%, 30/112). Three patients who gave false negative results all had enlarged nodes. Methylene blue is an effective tracer to detect SLNs in patients with early stage cervical cancer. The ideal dose and timing of methylene blue application are 4 ml and 60-90 min prior surgery, respectively.

Patent blue V and indocyanine green for fluorescence microimaging of human peritoneal carcinomatosis using probe-based confocal laser endomicroscopy.

PubMed

Abbaci, Muriel; Dartigues, Peggy; De Leeuw, Frederic; Soufan, Ranya; Fabre, Monique; Laplace-Builhé, Corinne

2016-12-01

Peritoneal carcinomatosis is a metastatic stage aggravating abdominal and pelvic cancer dissemination. The preoperative evaluation of lesions remains difficult today. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of tissue architecture and cellular details. This technology allows in vivo histological interpretation of tissue. The main limitation of pCLE for adoption in the clinic is the unavailability of fluorescent contrast agents. The aim of our study was to evaluate the staining performance of indocyanine green and patent blue V for histological diagnosis of pCLE images of pathological and non-pathological peritoneal tissue. We performed a correlative study with the histological gold standard on ex vivo human specimens from 25 patients operated for peritoneal carcinomatosis; 70 specimens were stained by topical application with ICG or patent blue V and then imaged with a probe-based confocal laser endomicroscope. A total of 350 pCLE images and 70 corresponding histological sections were randomly and blindly interpreted by two pathologists (PT1 and PT2). The images were first classified into two categories, tumoral versus non-tumoral, and a refined histological diagnosis was then given. All presented images were interpreted by PT1 (who received prior training on PCLE image reading) and PT2 (no training). 100 % sensitivity for PT1 and PT2 was noticed with tissues stained with ICG to differentiate tumoral and non-tumoral tissue. Global scores were always better for PT1 (major concordance between 86 and 94 %) than for PT2 (major concordance between 77 and 89 %) independently of the fluorescent dye when histological diagnosis was done on pCLE images. In conclusion, the pair ICG-pCLE offers the best combination for a non-trained pathologist for the interpretation of pCLE images from peritoneum.

[Technical recommendations and best practice guidelines for May-Grünwald-Giemsa staining: literature review and insights from the quality assurance].

PubMed

Piaton, Eric; Fabre, Monique; Goubin-Versini, Isabelle; Bretz-Grenier, Marie-Françoise; Courtade-Saïdi, Monique; Vincent, Serge; Belleannée, Geneviève; Thivolet, Françoise; Boutonnat, Jean; Debaque, Hervé; Fleury-Feith, Jocelyne; Vielh, Philippe; Cochand-Priollet, Béatrix; Egelé, Caroline; Bellocq, Jean-Pierre; Michiels, Jean-François

2015-08-01

May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Potential of 3-D tissue constructs engineered from bovine chondrocytes / silk fibroin-chitosan for in vitro cartilage tissue engineering

PubMed Central

Bhardwaj, Nandana; Nguyen, Quynhhoa T; Chen, Albert C; Kaplan, David L.; Sah, Robert L; Kundu, Subhas C

2011-01-01

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering. PMID:21601277

Characterization and Optimization of Bioflocculant Exopolysaccharide Production by Cyanobacteria Nostoc sp. BTA97 and Anabaena sp. BTA990 in Culture Conditions.

PubMed

Tiwari, Onkar Nath; Khangembam, Romi; Shamjetshabam, Minerva; Sharma, Aribam Subhalaxmi; Oinam, Gunapati; Brand, Jerry J

2015-08-01

Bioflocculant exopolysaccharide (EPS) production by 40 cyanobacterial strains during their photoautotrophic growth was investigated. Highest levels of EPS were produced by Nostoc sp. BTA97 and Anabaena sp. BTA990. EPS production was maximum during stationary growth phase, when nitrogenase activity was very low. Maximum EPS production occurred at pH 8.0 in the absence of any combined nitrogen source. The cyanobacterial EPS consisted of soluble protein and polysaccharide that included substantial amounts of neutral sugars and uronic acid. The EPS isolated from Anabaena sp. BTA990 and Nostoc sp. BTA97 demonstrated high flocculation capacity. There was a positive correlation between uronic acid content and flocculation activity. The flocculant bound a cationic dye, Alcian Blue, indicating it to be polyanionic. The 16S rRNA gene sequences for Nostoc sp. BTA97 and Anabaena sp. BTA990 were deposited at NCBI GenBank, and accession numbers were obtained as KJ830951 and KJ830948, respectively. The results of these experiments indicate that strains Anabaena sp. BTA990 and Nostoc sp. BTA97 are good candidates for the commercial production of EPS and might be utilized in industrial applications as an alternative to synthetic and abiotic flocculants.

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

PubMed

Hannesson, Kirsten O; Ytteborg, Elisabeth; Takle, Harald; Enersen, Grethe; Bæverfjord, Grete; Pedersen, Mona E

2015-08-01

In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

Shade changing effectiveness of plasdone and blue covarine-based whitening toothpaste on teeth stained with chlorhexidine and black tea.

PubMed

Bergesch, Vania; Baggio Aguiar, Flávio Henrique; Turssi, Cecilia Pedroso; Gomes França, Fabiana Mantovani; Basting, Roberta Tarkany; Botelho Amaral, Flávia Lucisano

2017-01-01

This study evaluated the effectiveness of toothbrushing with whitening toothpaste in altering the shade of stained human enamel. Thirty fragments of human enamel, stained with chlorhexidine/black tea underwent 1000 and 5000 brushing cycles (BC) with ( n = 10): PLS (Gel Dental Day, Bitufo), Close Up White Now, Unilever (COVB) and regular (Gel Dental Night, Bitufo) toothpaste. Images were taken before staining (baseline), after staining (STN) and following 1000 and 5000 BC and were analyzed using the CIELAB parameters (ΔE, Δb and ΔL). Data were submitted to two-way ANOVA (α = 0.05). ΔE was higher from STN to baseline; 1000 BC to STN and 5000 BC to STN ( P < 0.001). Significant differences in Δb values were found from 1000 BC to STN and 5000 BC to STN. For COVB, greater ΔL was observed from 1000 BC to STN, what differed statistically from the regular toothpaste ( P < 0.05). There was no difference between toothpaste when ΔL was calculated from 5000 CB to STN. Toothpaste containing COVB or PLS in association with 5000 BCs showed similar effectiveness in changing enamel shade; but after the first 1000 toothbrushing cycles, the use of COVB toothpaste promoted higher lightness in stained enamel.

Intraoperative Visualization of a Spinal Arachnoid Cyst Using Pyoktanin Blue.

PubMed

Takamiya, Soichiro; Seki, Toshitaka; Yamazaki, Kazuyoshi; Sasamori, Toru; Houkin, Kiyohiro

2018-01-01

Spinal arachnoid cysts (SACs) are filled with cerebrospinal fluid, and they include the arachnoid membrane, making it difficult to distinguish the walls of the cyst from the arachnoid membrane and excise the cyst as a lump. Here we report a technique for the intraoperative visualization of SACs, involving the use of pyoktanin blue. Four patients with spinal intradural arachnoid cysts underwent total excision of the cysts between October 2016 and April 2017. In 1 case, magnetic resonance imaging revealed the cyst clearly, but in the other cases, the cysts were unclear. All cysts were injected with 1% pyoktanin blue (Wako Pure Chemical Industries, Osaka, Japan) diluted 500 times with physiological saline before excision. When it was difficult to distinguish the cyst from the normal arachnoid membrane, 1% pyoktanin blue diluted 1000 times with physiological saline was injected into both the cyst and the subarachnoid space, and the spread of the stain was observed. The cysts were better visualized after pyoktanin blue injection than before injection. When it was difficult to distinguish the cyst from the normal arachnoid space, pyoktanin blue injection was useful for judging the cyst space. There were no perioperative complications, and the patients' symptoms improved partially or completely after treatment. Our technique of pyoktanin blue injection into SACs could make their excision easy and safe. Copyright © 2017 Elsevier Inc. All rights reserved.

Methylene Blue Assay for Estimation of Regenerative Re-Epithelialization In Vivo.

PubMed

Milyavsky, Maresha; Dickie, Renee

2017-02-01

The rapidity with which epithelial cells cover a wound surface helps determine whether scarring or scar-less healing results. As methylene blue is a vital dye that is absorbed by damaged tissue but not undamaged epidermis, it can be used to assess wound closure. We sought to develop a quantitative methylene blue exclusion assay to estimate the timeframe for re-epithelialization in regenerating appendages in zebrafish and axolotls, two classic model systems of regeneration. Following application of methylene blue to the amputation plane and extensive washing, the regenerating tail was imaged in vivo until staining was no longer visible. The percent area of the amputation plane positive for methylene blue, representing the area of the amputation plane not yet re-epithelialized, was measured for each time point. The loss of methylene blue occurred rapidly, within ~2.5 h in larval and juvenile axolotls and <1 h in adult zebrafish, consistent with high rates of re-epithelialization in these models of regeneration. The assay allows simple, rapid estimation of the time course for regenerative re-epithelialization without affecting subsequent regenerative ability. This technique will permit comparison of re-epithelialization across different strains and stages, as well as under the influence of various pharmacological inhibitors that affect regeneration.

A Synopsis of the Taxonomic Revisions in the Genus Ceratocystis Including a Review of Blue-Staining Species Associated with Dendroctonus Bark Beetles

Treesearch

Thelma J. Perry

1991-01-01

Taxonomic revisions in both the teleomorphic (sexual) and anamorphic (asexual) forms of the genus Ceratocystis Ellis & Halstead are chronicled in this review. Recognized species associated with Dendroctonus Erichson bark beetles are summarized, and several species that have been published as recombinations, species that were...

Bark beetle-induced tree mortality alters stand energy budgets due to water budget changes

Treesearch

David E. Reed; Brent E. Ewers; Elise Pendall; John Frank; Robert Kelly

2016-01-01

Insect outbreaks are major disturbances that affect a land area similar to that of forest fires across North America. The recent mountain pine bark beetle (Dendroctonus ponderosae) outbreak and its associated blue stain fungi (Grosmannia clavigera) are impacting water partitioning processes of forests in the Rocky Mountain region as the spatially heterogeneous...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2010 CFR

2010-01-01

... test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses. (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the... grass seed: (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2011 CFR

2011-01-01

... test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses. (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the... grass seed: (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind...

Photodynamic action of methylene blue in osteosarcoma cells in vitro.

PubMed

Guan, Jiemin; Lai, Xiaoping; Wang, Xinna; Leung, Albert Wingnang; Zhang, Hongwei; Xu, Chuanshan

2014-03-01

Osteosarcoma is a common malignant bone tumor which threatens the life of young people worldwide. To explore alternative strategy for combating osteosarcoma, a light-emitting diode (LED) that activates methylene blue (MB) was used in the present study to investigate cell death of osteosarcoma-derived UMR106 cells. Photocytotoxicity in UMR106 cells was investigated 24h after photodynamic activation of MB using sulforhodamine B (SRB) assay and light microscopy. Apoptosis induction was observed 24h after photodynamic treatment using a confocal laser scanning microscopy (CLSM) with Hoechst 33342 staining. The change in mitochondrial membrane potential (MMP) was analyzed using a flow cytometry with rhodamine 123 staining. MB under red light irradiation caused a drug-concentration (0-100μM) and light-dose (0-32J/cm(2)) dependent cytotoxicity in UMR106 cells. The SRB assay and light microscopy observed a significant decrease in the number of UMR106 cells attached to the bottom of culture well after LED light-activated MB (100μM, 32J/cm(2)). Nuclear shrinkage, chromatin condensation and fragmentation were found in the treated cells by nuclear staining. In addition, flow cytometry showed that the MMP in UMR106 cells was rapidly reduced by photo-activated MB (100μM, 32J/cm(2)). Photodynamic action of MB under LED irradiation could remarkably kill osteosarcoma cells and induce cell apoptosis as well as MMP collapse. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

New uses for calcium chloride solution as a mounting medium.

PubMed

Herr, J M

1992-01-01

Fresh cross sections of stems (Psilotum nudum, Coleus blumei, and Pelargonium peltatum) and roots (Setcreasea purpurea) 120 microns thick were fixed in FPA50 (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl2 solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.

Quantification of AAV particle titers by infrared fluorescence scanning of coomassie-stained sodium dodecyl sulfate-polyacrylamide gels.

PubMed

Kohlbrenner, Erik; Henckaerts, Els; Rapti, Kleopatra; Gordon, Ronald E; Linden, R Michael; Hajjar, Roger J; Weber, Thomas

2012-06-01

Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.

Notch3 is involved in adipogenesis of human adipose-derived stromal/stem cells.

PubMed

Sandel, Demi A; Liu, Mengcheng; Ogbonnaya, Ngozi; Newman, Jamie J

2018-07-01

Human adipose-derived stromal/stem cells (hASCs) have tremendous therapeutic potential and the ability to offer insight into human development and disease. Here we subject human ASCs to siRNA-mediated knockdown of Notch3 cultured under both self-renewing and adipogenic differentiation conditions. Self-renewal was monitored by assessing viability and proliferation rates through staining and alamarBlue assays, respectively. Adipogenesis was measured through Oil-Red O staining, western blot, and quantitative real-time RT-PCR that determined expression levels of multipotency and adipogenic markers over time. Notch3 was expressed in self-renewing hASCs but knockdown, as validated by qRT-PCR and western blot, showed no impact on cell viability, as measured through live-dead staining, or cell proliferation rates, as measured through alamarBlue assays. However, although Notch3 expression was observed to increase during adipogenesis, in the absence of Notch3 there was a significant increase in hASC adipogenesis as demonstrated through an increased number of lipid vesicles, and increased expression of adipogenic markers ppar-γ, adiponectin, fabp4, and plin2. Although Notch3 is only one of four Notch receptors expressed on the surface of hASCs, this receptor appears important for proper regulation of adipogenic differentiation, possibly serving as a negative regulator to prevent inappropriate adipogenesis or promote other lineage commitments of ASCs. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

NASA Technical Reports Server (NTRS)

Thompson, J. L.; Vijayan, K.; Riley, D. A.

2000-01-01

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523). Copyright 2000 Wiley-Liss, Inc.

Hue-saturation-density (HSD) model for stain recognition in digital images from transmitted light microscopy.

PubMed

van Der Laak, J A; Pahlplatz, M M; Hanselaar, A G; de Wilde, P C

2000-04-01

Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose. Copyright 2000 Wiley-Liss, Inc.

Assessment of the Dutch organ-culture system of corneal preservation within the Eye Bank of South Australia.

PubMed

Williams, K A; Noack, L M; Alfrich, S J; Danz, R; Erickson, S A; Coster, D J

1988-02-01

Thirteen per cent of all corneas harvested by the Eye Bank of South Australia during 1986 were discarded because storage time in McCarey-Kaufman medium exceeded four days. We have therefore examined the suitability of the Dutch method of long-term corneal storage for our purposes. Twenty-two human corneas that had been discarded from the Eye Bank were assessed using the trypan blue-sucrose staining technique, and then placed into long-term storage for 15 to 17 days. They were then reassessed by vital dye staining before permanent flat-mounts were prepared for silver staining of the endothelium. A good correlation (albeit subjective) was found between the non-destructive and destructive techniques of endothelial cell assessment. Those corneas that failed to survive organ culture storage were easily detected. The Dutch system of corneal preservation and post-storage assessment seems well-suited to Australian eye-banking.

Ultrastructural visualisation of proteoglycans in early unmineralised dentine of rat tooth germs stained with cuprolinic blue.

PubMed Central

Tenorio, D; Reid, A R; Katchburian, E

1990-01-01

The ultrastructural distribution and localisation of proteoglycans (PGs) of early developing rat dentine were examined using cuprolinic blue in a critical electrolyte concentration procedure. Results show that the cuprolinic blue method produces images of higher morphological quality than other cationic dyes. PGs appeared as ribbon-like electron-opaque precipitates of various sizes, ranging between 1.4 and 0.2 microns in length, distributed throughout the matrix and in close association with well preserved matrix vesicles and collagen fibrils. Matrix vesicles revealed tightly packed PG filaments which appeared to be attached to their membrane. It is possible that the close association of PG filaments with matrix vesicles and collagen indicates that PGs are related to the process of mineralisation of dentine. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:2384338

Disruption of the blood–brain barrier in pigs naturally infected with Taenia solium, untreated and after anthelmintic treatment

PubMed Central

Guerra-Giraldez, Cristina; Marzal, Miguel; Cangalaya, Carla; Balboa, Diana; Orrego, Miguel Ángel; Paredes, Adriana; Gonzales-Gustavson, Eloy; Arroyo, Gianfranco; García, Hector H.; González, Armando E.; Mahanty, Siddhartha; Nash, Theodore E.

2014-01-01

Neurocysticercosis is a widely prevalent disease in the tropics that causes seizures and a variety of neurological symptoms in most of the world. Experimental models are limited and do not allow assessment of the degree of inflammation around brain cysts. The vital dye Evans Blue (EB) was injected into 11 pigs naturally infected with Taenia solium cysts to visually identify the extent of disruption of the blood brain barrier. A total of 369 cysts were recovered from the 11 brains and classified according to the staining of their capsules as blue or unstained. The proportion of cysts with blue capsules was significantly higher in brains from pigs that had received anthelmintic treatment 48 and 120 h before the EB infusion, indicating a greater compromise of the blood brain barrier due to treatment. The model could be useful for understanding the pathology of treatment-induced inflammation in neurocysticercosis. PMID:23684909

The effect of saponification on the mucopolysaccharides of the ground substance of the human brain: the relation to focal edema and multiple sclerosis.

PubMed

Feigin, I

1981-03-01

The acid mucopolysaccharides of brain tissues are disclosed by their metachromatic staining with toluidine blue following saponification with potassium hydroxide, presumably as a result of the liberation of acid groups previously esterified. Earlier histochemical studies had disclosed the presence of neutral mucopolysaccharides by staining with the periodic acid-Schiff technique, and such staining is intensified by prior saponification. Many biochemical studies have reported the presence of both acid and neutral mucopolysaccharides in brain tissues. Within the white matter following brain edema, the quantity of stained mucopolysaccharides is decreased in the plaques of multiple sclerosis and pontine myelinolysis, and in the lesions of diffuse sclerosis. All of these are characterized by myelin loss with relative preservation of axons. The known physiological effects of the mucopolysaccharides on the water content of normal tissues, and on the properties and diffusability of the increments of fluid that constitute edema, lead to the suggestion that edema may play a major role in the pathogenesis of the demyelinating diseases, including multiple sclerosis.

Laparoscopic sentinel node procedure using a combination of patent blue and radiocolloid in women with endometrial cancer.

PubMed

Barranger, Emmanuel; Cortez, Annie; Grahek, Dany; Callard, Patrice; Uzan, Serge; Darai, Emile

2004-03-01

We assessed the feasibility of a laparoscopic sentinel node (SN) procedure based on the combined use of radiocolloid and patent blue labeling in patients with endometrial cancer. Seventeen patients (median age, 69 years) with endometrial cancer of stage I (16 patients) or stage II (1 patient) underwent a laparoscopic SN procedure based on combined radiocolloid and patent blue injected pericervically. After the SN procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopically assisted vaginal hysterectomy (16 patients) or laparoscopic radical hysterectomy (1 patient). SNs (mean number per patient, 2.6; range, 1-4) were identified in 16 (94.1%) of the 17 patients. Macrometastases were detected in three SNs from two patients by hematoxylin and eosin staining. In three other patients, immunohistochemical analysis identified six micrometastatic SNs and one SN containing isolated tumor cells. No false-negative SN results were observed. An SN procedure based on a combination of radiocolloid and patent blue is feasible in patients with early endometrial cancer. Combined use of laparoscopy and this SN procedure permits minimally invasive management of endometrial cancer.

Morphology and protein patterns of honey bee drone accessory glands.

PubMed

Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

2005-09-30

We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen.

Reconnaissance geology of the Al Mukhul Quadrangle, sheet 26/42 B, Kingdom of Saudi Arabia

USGS Publications Warehouse

Du Bray, E.A.

1984-01-01

Mineral potential in the quadrangle is low. At a very small prospect pit in the north-central part of the quadrangle, massive, milky quartz veins cutting weakly metamorphosed volcanogenic sedimentary rocks are stained blue and green by copper minerals. A previously reported mine site in the southern part of the quadrangle was not relocated.

Insect-caused deterioration of windthrown timber in northern California, 1963-1964

Treesearch

Boyd E. Wickman

1965-01-01

The principal tree degrade was caused by blue stain fungi introduced by bark beetles and flatheaded borers. Roundheaded borers caused the most serious wood boring. Smaller trees suffered more degrade than larger, and degrade was more severe below 5,000 feet elevation. Degrade due to insects was not as heavy as expected, probably because of cool summer weather and low...