Basis Of Staining Research Articles

Comparison of an Automated Pattern Analysis Machine Vision Time-lapse System with Traditional Endpoint Measurements in the Analysis of Cell Growth and Cytotoxicity

Machine vision is an application of computer vision. It both collects visual information and interprets the images. Although the machine obviously does not 'see' in the same sense that humans do, it is possible to acquire visual information and to create programmes to identify relevant image features in an effective and consistent manner. Machine vision is widely applied in industrial automation, but here we describe how we have used it to monitor and interpret data from cell cultures. The machine vision system used (Cell-IQ) consisted of an inbuilt atmosphere-controlled incubator, where cell culture plates were placed during the test. Artificial intelligence (AI) software, which uses machine vision technology, took care of the follow-up analysis of cellular morphological changes. Basic endpoint and staining methods to evaluate the condition of the cells, were conducted in parallel to the machine vision analysis. The results showed that the automated system for pattern analysis of morphological changes yielded comparable results to those obtained by conventional methods. The inbuilt software analysis offers a promising way of evaluating cell growth and various cell phases. The continuous follow-up and label-free analysis, as well as the possibility of measuring multiple parameters simultaneously from the same cell population, were major advantages of this system, as compared to conventional endpoint measurement methodology.

Relationship between Helicobacter pylori infection and expression of connexin (Cx) 32 and Cx43 genes in gastric cancer and gastric precancerous lesions

To investigate the expression of connexin (Cx)32 and Cx43 genes in gastric cancer and precancerous lesion, and to investigate the relation between the changes of expression of Cx32 and Cx43 genes and Helicobacter pylori (Hp) infection. Gastroscopy and biopsy of gastric mucosa were conducted on 33 patients with chronic superficial gastritis (CSG), 88 with precancerous lesion, and 70 with gastric cancer. Hp was detected by rapid urease test, basic fuchsin staining, and 14C-urea breath test. The CagA gene of Hp was determined by PCR. SABC immunohistochemical method was used to detect the expression of Cx32 and Cx43 genes in gastric mucosa biopsy specimens. The positive expression rates of Cx32 and Cx43 genes were 15.7% and 32.9% respectively in the gastric cancer patients, 51.1% and 54.5% in the patients with precancerous lesion, and 100.0% and 93.9% in the CSG patients. The positive Cx32 and Cx43 expression rates of the gastric cancer and precancerous lesion patients were significantly lower than those of the CSG patients (all P < 0.05). The positive Cx32 expression rate of the gastric cancer patients with Hp infection was 16.7%, not significantly different from that of the gastric cancer patients without Hp infection (13.6%). The positive Cx43 expression rate of the gastric cancer patients with Hp infection was 25%, significantly lower than that of the gastric cancer patients without Hp infection (50%, P = 0.039). The positive Cx32 and Cx43 expression rates and expression intensity of the precancerous lesion patients with Hp infection were all significantly lower than those of the precancerous lesion patients without Hp infection (all P < 0.05). The positive Cx43 expression rate of the gastric cancer patients with CagA+ Hp infection was 17.9%, significantly lower than that of the CagA- Hp group (55.6%, P = 0.027), however, the positive Cx32 expression rate of the gastric cancer patients with CagA+ Hp infection was 12.8%, not significantly different from that of the gastric cancer patients with CagA- Hp infection (33.3%, P = 0.159). The positive Cx32 and Cx43 expression rates of the CSG patients with CagA+ Hp and CagA- Hp infection were all 100%, but the expression intensity of the CagA+ Hp group was significantly lower than that of the CagA- Hp group (P = 0.032). The positive Cx32 and Cx43 expression rates after Hp eradication of the precancerous lesion patients with Hp infection were 97.9% and 91.7% respectively, both significantly higher than those before eradication therapy (41.4% and 44.8% respectively, both P < 0.05). However, the positive Cx32 and Cx43 expression rates of the precancerous lesion patients with Hp infection without Hp eradication were still 40% and 50%, not significantly different from those before treatment. The expression levels of Cx32 and Cx43 in gastric cancer and precancerous lesion decrease. The change of expression of Cx43 was associated with Hp infection, especially CagA+ Hp infection. Hp eradication in patients with precancerous lesion up-regulates the expression of Cx32 and Cx43.

RANKL Is a Mediator of Bone Resorption in Idiopathic Hypercalciuria

This study aimed to determine the expression of osteoprotegerin, receptor activator of nuclear factor kappaB ligand, interleukin-1alpha, transforming growth factor-beta, and basic fibroblast growth factor in stone-forming patients with idiopathic hypercalciuria. Immunohistochemical analysis was performed in undecalcified bone samples previously obtained from 36 transiliac bone biopsies of patients who had idiopathic hypercalciuria and whose histomorphometry had shown lower bone volume, increased bone resorption, and prolonged mineralization lag time. Bone expression of receptor activator of nuclear factor kappaB ligand and osteoprotegerin was significantly higher in patients with idiopathic hypercalciuria versus control subjects. Transforming growth factor-beta immunostaining was lower in patients with idiopathic hypercalciuria than in control subjects and correlated directly with mineralization surface. Interleukin-1alpha and basic fibroblast growth factor staining did not differ between groups. Receptor activator of nuclear factor kappaB ligand bone expression was significantly higher in patients who had idiopathic hypercalciuria and exhibited higher versus normal bone resorption. A higher expression of receptor activator of nuclear factor kappaB ligand in bone tissue suggests that increased bone resorption in patients with idiopathic hypercalciuria is mediated by receptor activator of nuclear factor kappaB ligand. Osteoprotegerin bone expression might have been secondarily increased in an attempt to counteract the actions of receptor activator of nuclear factor kappaB ligand. The low bone expression of transforming growth factor-beta could contribute to the delayed mineralization found in such patients.

Mandible Matrix Necrosis in Beagle Dogs After 3 Years of Daily Oral Bisphosphonate Treatment

An increasing number of reports have implicated bisphosphonates as contributing to osteonecrosis of the jaw. The goal of this study was to evaluate mandible necrosis in beagle dogs treated for 3 years with oral alendronate (ALN). Skeletally mature female beagles were treated daily for 3 years with oral doses of vehicle (VEH) or ALN (0.20 or 1.0 mg/kg/day). These doses approximate, on a mg/kg basis, those used for postmenopausal osteoporosis and Paget's disease, respectively. At necropsy, the second molar region of the mandible was excised, stained en bloc with basic fuchsin, and assessed for matrix necrosis and intracortical bone turnover rate using histology. Matrix necrosis was defined as a region greater than 500 microm(2) that was void of basic fuchsin stain, assessed using both bright-field and confocal microscopy. No animals developed exposed bone lesions in the oral cavity during the 3-year study. Matrix necrosis was observed in 25% of ALN0.2 animals, 33% of ALN1.0 animals, and was noticeably absent from all vehicle animals (P < .05 pooled ALN doses vs VEH). These necrotic regions occurred predominately in the alveolar bone and were clearly void of patent canaliculi. Intracortical bone turnover rate of the alveolar mandible bone region was significantly lower (-75%, P < .05) in ALN-treated animals compared with VEH. Three years of daily oral bisphosphonate treatment reduces bone turnover significantly and increases the incidence of matrix necrosis within the mandible of dogs.

Chromoendoscopy with hematoxylin in the classificaton of gastric lesions

Hematoxylin is a basic histological stain which stains nuclei. Apparently, it can be used with endoscopy to diagnose malignant lesions due to their increased nucleus-tocytoplasm ratio. The aim of this study was to evaluate the use of hematoxylin in the differentiation of gastric lesions. Part 1. In a prospective study, a total of 70 different gastric abnormalities were stained with 1% hematoxylin solution during upper endoscopy. Results of histological examinations were compared with endoscopic findings. Part 2. During a 5-day period, antral mucosa was stained with hematoxylin in all patients (n = 26), independent of the upper endoscopy results. Grades of stained mucosa were compared with the proliferating cell nuclear antigen (PCNA) index, which was examined using an immunohistochemical method. Part 3. Gastric biopsies (total, 12) were obtained from the stained and unstained areas within the limits of one lesion (total, 6) after chromoscopy with hematoxylin and examined with monoclonal (PCNA) antibodies. Using histological findings, the sensitivity and specificity of chromoendoscopy with hematoxylin for detecting gastric neoplasia and adenocarcinoma were determined. A significant positive correlation (r = 0.81; P < 0.001) was found between the PCNA index and grade of endoscopic stained mucosa. The PCNA index was increased in stained areas of gastric abnormalities in comparison to unstained areas within the limits of one lesion in all six patients. Chromoendoscopy with hematoxylin increases opportunities for precision biopsy. The grade of endoscopic stained mucosa depends on epithelial cell proliferation and is associated with malignancy. By using histology as the main outcome, the sensitivity and specificity of chromoendoscopy with hematoxylin for diffuse staining in the detection of gastric neoplasia and adenocarcinoma were 92.9% and 89.3%, respectively.

Hyperoxia Causes Maturation-Dependent Cell Death in the Developing White Matter

Periventricular leukomalacia is the predominant injury in the preterm infant leading to cerebral palsy. Oxygen exposure may be an additional cause of brain injury in these infants. In this study, we investigated pathways of maturation-dependent oligodendrocyte (OL) death induced by hyperoxia in vitro and in vivo. Developing and mature OLs were subjected to 80% oxygen (0-24 h). Lactate dehydrogenase (LDH) assay was used to assess cell viability. Furthermore, 3-, 6-, and 10-d-old rat pups were subjected to 80% oxygen (24 h), and their brains were processed for myelin basic protein staining. Significant cell death was detected after 6-24 h incubation in 80% oxygen in pre-OLs (O4+,O1-), but not in mature OLs (MBP+). Cell death was executed by a caspase-dependent apoptotic pathway and could be blocked by the pan-caspase inhibitor zVAD-fmk. Overexpression of BCL2 (Homo sapiens B-cell chronic lymphocytic leukemia/lymphoma 2) significantly reduced apoptosis. Accumulation of superoxide and generation of reactive oxygen species (ROS) were detected after 2 h of oxygen exposure. Lipoxygenase inhibitors 2,3,5-trimethyl-6-(12-hydroxy-5-10-dodecadiynyl-1,4-benzoquinone and N-benzyl-N-hydroxy-5-phenylpentamide fully protected the cells from oxidative injury. Overexpression of superoxide dismutase (SOD1) dramatically increased injury to pre-OLs but not to mature OLs. We extended these studies by testing the effects of hyperoxia on neonatal white matter. Postnatal day 3 (P3) and P6 rats, but not P10 pups, showed bilateral reduction in MBP (myelin basic protein) expression with 24 h exposure to 80% oxygen. Hyperoxia causes oxidative stress and triggers maturation-dependent apoptosis in pre-OLs, which involves the generation of ROS and caspase activation, and leads to white matter injury in the neonatal rat brain. These observations may be relevant to white matter injury observed in premature infants.

Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

IntroductionTo understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations.MethodsFreshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an α-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines.ResultsAnalysis of the separated populations demonstrated that Sca-1- 33A10High stained cells were estrogen receptor α (Esr1)- luminal epithelial cells, whereas Sca-1+ 33A10Low/- stained cells were a mix of nonepithelial cells and Esr1+ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1- 33A10Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi-quantitative RT-PCR analysis, data mining and quantitative real-time RT-PCR approaches, which showed that RGS2 was expressed in the majority of solid breast cancers at much higher levels than in normal human mammary cells.ConclusionMolecular analysis of prospectively isolated mammary epithelial cells identified RGS2 as a modulator of oxytocin receptor signalling, which is highly expressed in the myoepithelial cells. The RGS2 gene, but not the oxytocin receptor, was also shown to be over-expressed in the majority of breast cancers, identifying the product of this gene, or the pathway(s) it regulates, as potentially significant therapeutic targets.

The TLC: A Novel Auditory Nucleus of the Mammalian Brain

We have identified a novel nucleus of the mammalian brain and termed it the tectal longitudinal column (TLC). Basic histologic stains, tract-tracing techniques and three-dimensional reconstructions reveal that the rat TLC is a narrow, elongated structure spanning the midbrain tectum longitudinally. This paired nucleus is located close to the midline, immediately dorsal to the periaqueductal gray matter. It occupies what has traditionally been considered the most medial region of the deep superior colliculus and the most medial region of the inferior colliculus. The TLC differs from the neighboring nuclei of the superior and inferior colliculi and the periaqueductal gray by its distinct connections and cytoarchitecture. Extracellular electrophysiological recordings show that TLC neurons respond to auditory stimuli with physiologic properties that differ from those of neurons in the inferior or superior colliculi. We have identified the TLC in rodents, lagomorphs, carnivores, nonhuman primates, and humans, which indicates that the nucleus is conserved across mammals. The discovery of the TLC reveals an unexpected level of longitudinal organization in the mammalian tectum and raises questions as to the participation of this mesencephalic region in essential, yet completely unexplored, aspects of multisensory and/or sensorimotor integration.

The Giemsa Stain: Its History and Applications

Gustav Giemsa was born in Germany in 1867, worked mainly as a chemist, and died in 1948. The staining method, which carries his name, was designed primarily for the demonstration of parasites in malaria, but it was also employed in histology because of the high-quality staining of the chromatin and the nuclear membrane, the metachromasia of some cellular components, and the different qualities of cytoplasmic staining depending on the cell type. The use of methylene azure and its mixture with methylene blue to form an eosinate made stable the stain and its results. Giemsa's stain is regarded as the world's standard diagnostic technique for malaria's plasmodium, and it is also the basic stain for classifying lymphomas in the Kiel classification.

Correlation of Confocal Endomicroscopy Imaging with Immunohistochemical Localization Patterns of Fluorescein in Biopsies of Colon Polyps and Cancers

Background: Confocal endomicroscopy (CEM) is a newly developed system that uses a laser confocal system together with a conventional endoscope to provide additional digital information about the tissue surface, subsurface morphology and microstructure. In our previous study, we reported that the localization of injected fluorescein was consistent with the signal in the CEM images and the CEM might be an important device for virtual biopsy. In this study, we evaluated CEM as a tool for conducting virtual biopsies of the colon by comparing structure and architecture in CEM images with biopsy histopathology of neoplastic lesions including hyperplastic polyps, adenomatous polyps and colon cancers. Methods: The large intestines of 51 patients (35 males and 16 females), with an average age of 64 years (range, 37 to 76 years) were examined using an intravenous injection of fluorescein during CEM to facilitate correlation of images taken in vivo with biopsied samples of each lesion. Six hyperplastic polyps, 12 adenomous polyps, and 13 colonic cancers were evaluated. CEM images were compared with conventional histopathology of biopsies. For correlation of biopsy sites corresponding to CEM images, fluorescein injected at endoscopy was localized immunohistochemically with the avidin-biotin complex (ABC) immunoperoxidase technique using mouse monoclonal antibody to fluorescein as the primary antibody. Results: All CEM images were concordant histopathologically with biopsy observations. Fluorescein immunohistochemical staining of both hyperplastic and adenomous polyps was the same as normal colon tissue. That is, staining is localized in the interstitium, capillary walls and at the mucosal surface of crypts. The mucus of goblet cells and the cytoplasm of mucosal surface enterocytes were stained, but not their nuclei. These immunohistochemical patterns were consistent with the fluorescein localization patterns in the CEM images. The stain distribution within colon cancers was similar to that in polyps, but stain localization was very irregular in pattern and intensity. Conclusions: All CEM images were consistent with their histopathological counterparts. In the hyperplastic and adenomous lesions the fluorescein localization corresponded to the CEM images and the distribution was the same as in normal colon tissue. The fluorescein localization in cancer showed irregular pattern and variable intensity, though the basic staining distribution was the same as in the other lesions. These observations further validate the utility of CEM for in vivo assessment of colonic lesions.

P-01 Hormones and Sexuality

The infection process was compared in interactions between Erysiphe graminis f.sp. hordei isolate A6 and two near-isogenic barley lines (the susceptible line P-01 and the resistant line P-02), and the non-host wheat. A low frequency of successful infections, measured as the percentage of germinated conidia forming primary haustoria, was detected in both susceptible (7·5%), and resistant (73%) barley isolines and wheat plants (223%). In all three types of plants, papilla formation around the sites of attempted penetration seems to be a general resistance factor and thus not the determining step for distinguishing between race-specific and non-host resistance. In wheat plants, further resistance was manifested early after penetration and characterized by hypersensitively reacting (HR) single cells, no or limited growth of elongating secondary hyphae and no visible sign of infection. The race-specific resistance of barley isoline P-02 occurred later after penetration, and was characterized by plant tissue necrosis and no or weak fungal sporulation. In the susceptible barley isoline P-01, successful infections resulted in heavily sporulating powdery mildew colonies. Putative guanidine compounds, indicated by the Sakaguchi test, occurred in papillae 4–5 h after inoculation and subsequently in HR cells too, and are apparently associated with the very early resistance responses in barley and wheat. The highly basic guanidine compounds, not identified previously in plant defence responses, were found to be bound to the cell wall, and referred to as “basic staining material”. Accumulation of polyphenolic substances and callose were demonstrated in papillae and HR cells of both barley and wheat. Lignification occurred in wheat, but did not occur or did so only minimally in barley.

Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme

Adenosine is a potent modulator of excitatory neurotransmission, especially in seizure-prone regions such as the hippocampal formation. In adult brain ambient levels of adenosine are controlled by adenosine kinase (ADK), the major adenosine-metabolizing enzyme, expressed most strongly in astrocytes. Since ontogeny of the adenosine system is largely unknown, we investigated ADK expression and cellular localization during postnatal development of the mouse brain, using immunofluorescence staining with cell-type specific markers. At early postnatal stages ADK immunoreactivity was prominent in neurons, notably in cerebral cortex and hippocampus. Thereafter, as seen best in hippocampus, ADK gradually disappeared from neurons and appeared in newly developed nestin- and glial fibrillary acidic protein (GFAP)-positive astrocytes. Furthermore, the region-specific downregulation of neuronal ADK coincided with the onset of myelination, as visualized by myelin basic protein staining. After postnatal day 14 (P14), the transition from neuronal to astrocytic ADK expression was complete, except in a subset of neurons that retained ADK until adulthood in specific regions, such as striatum. Moreover, neuronal progenitors in the adult dentate gyrus lacked ADK. Finally, recordings of excitatory field potentials in acute slice preparations revealed a reduced adenosinergic inhibition in P14 hippocampus compared with adult. These findings suggest distinct roles for adenosine in the developing and adult brain. First, ADK expression in young neurons may provide a salvage pathway to utilize adenosine in nucleic acid synthesis, thus supporting differentiation and plasticity and influencing myelination; and second, adult ADK expression in astrocytes may offer a mechanism to regulate adenosine levels as a function of metabolic needs and synaptic activity, thus contributing to the differential resistance of young and adult animals to seizures.

Staining Polymers for Microscopical Examination

Abstract Polymers find widespread use in blends, graft copolymers, polymer alloys, and composites. Staining is an important tool for microscopically visualizing polymer phases and morphologies in these multi-component systems. Three stains have been used in most investigations. These stains (and the functional groups they are aimed at) are: osmium tetroxide for chemical unsaturation; ruthenium tetroxide for styrenics; and phosphotungstic acid for polyamides. Beyond these three basic stains there are a significant number of additional ones that have been developed for special cases. Aside from reacting chemically with functional groups, stains have been used as: negative (outline) stains; stains to delineate amorphous-crystalline regions; stains for microvoids, and stains for phase boundaries. Stains are most commonly transported to the target functional groups in tow ways — dissolved in liquid media or by direct contact with stain vapors. Improvisations have been developed to meet special staining problems. These improvisations most often involve: 1) chemical alteration of a polymer backbone to introduce a stainable functionality; 2) tailoring of staining media to provide adequate diffusion into the matrix; 3) preferential swelling of a blend component in order to accept a stainable moiety; 4) tactical use of a staining temperature to take advantage of differences in glass transition temperatures of blend components; and 5) optimization of chemical equilibria to expedite staining time or extent. Examples of these modifications are presented along with some cautionary notes regarding some artifact that may arise. A plea is made for more adequate descriptions of staining protocols in published articles. In too many cases there is inadequate description of staining methods and experimental duplication is put in jeopardy.

Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.

Evidence for specific phases in the development of human neuromelanin

Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. Here we describe the age-related development and regulation of neuromelanin within these dopamine neurons. 10 microm sections from formalin-fixed midbrain from 29 people spanning the ages of 24 weeks to 95 years old were either stained with a basic Nissl substance stain (0.5% cresyl violet), or processed unstained. After locating the substantia nigra using the stained sections, digital photos were taken of individual ventral substantia nigra neurons in the unstained sections, and the cellular area occupied by pigment, and optical density were measured using computer software. These measurements demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.

Proteomics reveals elevated levels of PR 10 proteins in saline-tolerant peanut ( Arachis hypogaea) calli

The proteome of a salinity-tolerant Arachis hypogaea L. callus cell line was compared with its sensitive counterpart. Several low molecular weight proteins were detected by two-dimensional electrophoresis as being unique or significantly elevated in the tolerant line. The identities of several of these proteins were established as PR 10 proteins using tandem Mass Spectrometry and are shown to be phosphorylated on the basis of staining with the phosphorylation-specific stain, Pro-Q Diamond. Our results suggest that these differentially phosphorylated PR 10 proteins may play an important role in mediating salinity stress responses.

Microscopic anatomy of the thin‐walled vessels leaving the heart of the lobsterHomarus americanus:anterior lateral arteries

Abstract.The anterior lateral arteries are paired vessels leaving the anterior end of the lobster (Homarus americanus) heart and proceeding to the antennae and eyestalks, the stomach and hepatopancreas, the gonads, and the thoracic and branchial muscles. These vessels have a trilaminar organization, consisting of a tunica interna with elastic fibrils, a tunica intermedia represented by a bilayered cell mass, and a tunica externa with collagen fibrils. In the tunica intermedia, cells flanking the tunica interna (light cells) show less affinity for basic dyes and electron stains than those flanking the tunica externa (dark cells). Each light cell exhibits an irregularly shaped stress fiber (a bundle of closely packed microfilaments) in the region adjoining the tunica interna. Collectively, these bundles have a circumferential or slightly oblique orientation relative to the lumen of the vessel. The role of the stress fibers is unresolved. If they are static structures, they might contribute to the non‐linear elasticity shown by lobster arteries. If they generate force, and small bundles of microfilaments do diverge from the stress fibers to enter filamentous mats applied to the plasmalemmata, a coordinated contraction of the cells might reduce the luminal diameter and, thus, retard the flow of hemolymph. Coordination of contraction would have to occur in the absence of nerves and without the benefit of communicating (gap) junctions between the light and dark cells.

Alcohol Abuse Enhances Neuroinflammation and Impairs Immune Responses in an Animal Model of Human Immunodeficiency Virus-1 Encephalitis

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Maturational Effects of Lipopolysaccharide on White-Matter Injury in Fetal Sheep

White-matter damage has been associated with the development of cerebral palsy in children born both prematurely and at term, and it has been suggested that intrauterine infection can contribute to the brain injury. However, the relative importance of age on white-matter injury following infectious exposure in utero remains unclear. In this study, fetal sheep were exposed to systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (88.7 +/- 7.7 ng/kg) at 65% or 85% of gestation. These gestational ages approximately correspond to human brain development in preterm and near-term infants respectively. White-matter injury was evaluated 3 days after lipopolysaccharide exposure with regard to microglia activation and loss of neurofilament and myelin basic protein. The expression of oligodendrocytes at different maturational stages was demonstrated in preterm and near-term fetuses with the oligodendroglial markers O4 and 2 ,3 -cyclic nucleotide 3 -phospodiesterase. Forty percent of the fetuses in the preterm group and 22% in the near-term group died within 8 hours of the endotoxin exposure. Three of six preterm and two of seven near-term surviving fetuses demonstrated pathologic changes in the brain with regard to increased microglia activation and loss of neurofilament staining. The number of activated microglia was enhanced in the subcortical white matter in both the preterm lipopolysaccharide-exposed fetuses (lipopolysaccharide: 235 +/- 64 cells/mm2; control: 72 +/- 28 cells/mm2; P = .0374) and the near-term fetuses (lipopolysaccharide: 180 +/- 40 cells/mm2; control 23 +/- 16 cells/mm2; P = .0152). There was a loss of neurofilament staining in both preterm fetuses (lipopolysaccharide: 2.20 +/- 0.77 pixel units; control: 0.20 +/- 0.10 pixel units; P = .0306) and near-term fetuses (lipopolysaccharide: 1.15 +/- 0.48 pixel units; control: 0.06 +/- 0.06 pixel units; P = .0285). O4-positive cells were detected at both gestational ages, whereas 2,3-cyclic nucleotide 3-phospodiesterase-positive cells and myelin basic protein staining were mainly detected in the near-term fetuses. In summary, we found white-matter injury in a proportion of both preterm and near-term fetuses after administration of lipopolysaccharide. These results are in agreement with clinical evidence suggesting that both preterm and term infants are at risk of periventricular leukomalacia in association with intrauterine infection.

Color matching algorithms in ceramic tile production

In the present work, the possibility of transferring in the ceramic tiles production the know-how developed in the field of the paints by using the Kubelka–Munk theory, in the form used for opaque surface coatings, have been evaluated. Five different target colors have been chosen as target and tried to reproduce with an industrial glaze in a cycle for fine porcelain stoneware tiles. Four industrial pigments have been chosen as basic stains for the formulations. The results show a good efficiency of the color matching algorithm applied to pigments for glazes for fine porcelain stoneware tiles. All the formulations, in fact, have allowed to obtain a value of Δ E * lower to the accepted limit.