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A procedure is described for analyzing total lipid (TL) and triacylglycerol (TAG) in 2 sequential steps using small amounts ( Key words: broken-line model, cattle, fatty liver, liver biopsy, total lipid, triacylglycerol doi: 10.2527/jas.2009-2599

Active [Na.sup.+] absorption across rumen epithelium comprises [Na.sup.+]/[H.sup.+] exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate [Na.sup.+] absorption, which has been attributed to an interaction between [Cl.sup.-]/HC[O.sup.-.sub.3] and [Na.sup.+]/[H.sup.+] exchangers. However, isolated rumen epithelial cells also express a [Cl.sup.-] conductance. We investigated whether [Cl.sup.-] has an additional effect on electrogenic [Na.sup.+] absorption via NSCC. NSCC was estimated from short-circuit current ([I.sub.sc]) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (p[H.sub.s]) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, [Cl.sup.-] stimulated the [Ca.sup.2+] and [Mg.sup.2+] sensitive [I.sub.sc]. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 [+ or -] 0.03 in a low-[Cl.sup.-] solution. It was increased by 0.21 pH units when luminal [Cl.sup.-] was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the [Ca.sup.2+] and [Mg.sup.2+] sensitive [I.sup.sc] and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal [Cl.sup.-] depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive [I.sub.sc], but only in low-[Cl.sup.-] solutions. The results show that luminal [Cl.sup.-] can increase the microclimate pH via apical [Cl.sup.-]/HC[O.sup.-.sub.3] or [Cl.sup.-]/O[H.sup.-] exchangers. Electrogenic [Na.sup.+] absorption via NSCC increases with pH, explaining part of the [Cl.sup.-] effects on [Na.sup.+] absorption. The data further show that the [Cl.sup.-] conductance of rumen epithelium must be located at the basolateral membrane. electrolyte transport; sodium absorption; forestomach; ruminants; microclimate doi:10.1152/ajpgi.00419.2005

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 ± 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane. [ABSTRACT FROM AUTHOR]

Butyrate exerts a trophic effect on the colonocytes and plays a protective role in ulcerative colitis. In the present study, we investigated the effect of butyrate withdrawal on the colonic mucosa of the guinea-pig. The samples were mounted in Ussing chambers and bathed for 45, 60, 90 and 150 min with standard Ringer solution with or without sodium butyrate. Light and electron microscopy for morphology, electrophysiological methods for testing tissue function, histochemistry using the TUNEL method for localization of apoptotic cells and flow cytometry for cell cycle analysis were applied. Morphological observations revealed that butyrate deprivation caused a time-dependent hypoplasia and a rapid triggering of massive apoptosis as substantiated by the TUNEL assay. The epithelium, however, did not show discontinuities at any time. Electrophysiological data confirmed that no leakage of the epithelium had occurred. In the control specimens, the mucosa underwent a moderate reduction in height; apoptotic epithelial cells were infrequently observed. Cell cycle analysis of co-lonocytes isolated from the mucosa deprived of butyrate revealed a decrease in the percentage of cells occupying each phase of the cycle, especially the G/Gphase. Thus, in the absence of butyrate, apoptosis was enhanced and cell renewal reduced. The trophic protective action exerted by butyrate in both physiological and pathological conditions could derive from its capacity to modulate survival and death of colonocytes. [ABSTRACT FROM AUTHOR]

According to the World Health Organization (WHO), around 60% of all outbreaks are detected using informal sources. In many public health institutes, including the WHO and the Robert Koch Institute (RKI), dedicated groups of public health agents sift through numerous articles and newsletters to detect relevant events. This media screening is one important part of event-based surveillance (EBS). Reading the articles, discussing their relevance, and putting key information into a database is a time-consuming process. To support EBS, but also to gain insights into what makes an article and the event it describes relevant, we developed a natural language processing framework for automated information extraction and relevance scoring. First, we scraped relevant sources for EBS as done at the RKI (WHO Disease Outbreak News and ProMED) and automatically extracted the articles' key data: disease, country, date, and confirmed-case count. For this, we performed named entity recognition in two steps: EpiTator, an open-source epidemiological annotation tool, suggested many different possibilities for each. We extracted the key country and disease using a heuristic with good results. We trained a naive Bayes classifier to find the key date and confirmed-case count, using the RKI's EBS database as labels which performed modestly. Then, for relevance scoring, we defined two classes to which any article might belong: The article is relevant if it is in the EBS database and irrelevant otherwise. We compared the performance of different classifiers, using bag-of-words, document and word embeddings. The best classifier, a logistic regression, achieved a sensitivity of 0.82 and an index balanced accuracy of 0.61. Finally, we integrated these functionalities into a web application called EventEpi where relevant sources are automatically analyzed and put into a database. The user can also provide any URL or text, that will be analyzed in the same way and added to the database. Each of these steps could be improved, in particular with larger labeled datasets and fine-tuning of the learning algorithms. The overall framework, however, works already well and can be used in production, promising improvements in EBS. The source code and data are publicly available under open licenses., Competing Interests: The authors have declared that no competing interests exist.

We studied the esophageal epithelium for keratinization characteristics from samples of domesticated mammals of three nutrition groups (herbivores: horse, cattle, sheep; omnivores: pig, dog, rat; carnivores: cat) using histochemistry (keratins, disulfides), sulfur measurements, and cryo-SEM. Keratins were found in all esophageal layers of all species, except for the equine Stratum corneum. The positive reaction staining of Pan-keratin was remarkable, but decreased in intensity toward the outer layers, whereas in the pig and cat, staining was confined to the corneal layer. The herbivores revealed positive staining reactions in the upper Stratum spinosum, particularly in the sheep. Regarding single keratins, CK6 immunostating was found in most esophageal layers, but only weakly or negatively in the porcine and equine Stratum corneum. CK13 staining was restricted to the sheep and here was found in all layers. CK14 could be detected in the equine and feline Stratum basale, and upper vital layers of the dog and rat. CK17 appeared only in the Stratum spinosum and Stratum granulosum, but in all layers of the dog and cat. Disulfides reacted strongest in the Stratum corneum of the herbivores, as corroborated by the sulfur concentrations in the esophagus. Our study emphasized that keratins are very important for the mechanical stability of the epithelial cells and cell layers of the mammalian esophagus. The role of these keratins in the esophageal epithelia is of specific interest owing to the varying feed qualities and mechanical loads of different nutrition groups, which have to be countered., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The sensitive red fluorescence dye BODIPY® 665/676, and embedding in the water-soluble resin Technovit® 7100 were used to demonstrate free fatty acids in the epidermis of seven semi-aquatic and aquatic mammalian species with a sparse or dense hair coat. The staining generally marked lipid layers of varying thickness between the lamellae of the Stratum corneum, as found particularly in very densely haired species (otter), but also in rather sparsely haired animals (beaver, nutria), and especially in the seal. The very sparsely haired capybara contained no free fatty acids in the corneal layer system, but exhibited an accumulation of such substances in the vital epidermis. All haired species showed a strongly positive reaction staining of the sebaceous glands. In the hairless species, a distinct intracellular staining was restricted to cells of the thick vital epidermis in the hippopotamus, whereby in the Str. corneum positive intercellular reactions appeared. In the dolphin, on the contrary, positive intercellular reactions became visible in the vital epidermis, whereas in the Str. corneum the lipids concentrated in large longitudinal intracellular vesicles., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The study demonstrates the free sugar spectrum of the secretion of the tubuloaveolar temporal gland of the African elephant (Loxodonta africana), using lectin histochemistry. In the elephant, the spectrum contained, besides strongly varying amounts of α-D-mannose, very remarkable reactions for α-D-galactose and to a certain extent also for α-D-N-acetyl-galactosamine or a/ß-D-N-acetylglucosamine. This is in contrast to the free sugar spectrum of the secretion of the mammalian apocrine tubular skin glands. Considering also that the production of any water binding mucus seems to be negligible, the variations of the free sugar contents found probably originate from another important task of the secretory cells of the temporal gland. This means that our findings corroborate the view of highly active acquired immunity by an intensive processing and presenting of lipid antigens by dendritic cells (APC), in particular of the CD1 family.

A neutral pH microclimate had been shown at the luminal surface of the large intestine. The aim was to estimate to what extent fluxes of propionic acid/propionate are affected by changes of the luminal pH when this microclimate is present, largely reduced or absent. Fluxes of propionic acid/propionate (J(Pr)) across epithelia from the caecum, the proximal and the distal colon of guinea pigs were measured in Ussing chambers with and without a filter at the luminal surface. With bicarbonate and with a neutral or an acid pH of mucosal solutions (pH 7.4 or 6.4), mucosal-to-serosal fluxes (J(ms)(Pr) ) were 1.5 to 1.9-fold higher at the lower pH, in bicarbonate-free solutions and carbonic anhydrase (CA) inhibition 2.1 to 2.6-fold. With a filter at the mucosal surface and with bicarbonate containing solutions, J (ms) (Pr) was not or only little elevated at the lower pH. Without bicarbonate J(ms)(Pr) was clearly higher. We conclude that the higher J(ms)(Pr) after luminal acidification is due to vigorous mixing in Ussing chambers resulting in a markedly reduced unstirred layer. Therefore, an effective pH microclimate at the epithelial surface is missing. J(ms)(Pr) is not or is little affected by lowering of pH because in the presence of bicarbonate the filter maintains the pH microclimate. However, in bicarbonate-free solutions J(ms)(Pr) was higher at pH 6.4 because a pH microclimate does not develop. Findings confirm that 30-60% of J(ms)(Pr) results from non-ionic diffusion.

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.

Biochemical, histochemical and cytochemical analyses were used to determine the sulphur contents and the thiol and disulphide distribution in the stratum corneum (SC) of the wild boar (WB), a large domestic pig breed (DP) and the Goettingen miniature pig (GMP). The sulphur contents (% DW) were different in the three animal types (WB: 1.70-1.38 body, 0.54 ear; DP: 0.84-0.53 body, 0.50 ear; GMP: 2.28-2.51 body, 2.66 ear). The results of the histochemical analysis of SH- and -S-S- groups were clear, and densitometrical extinctions were highest in most body regions of the GMP for thiols and disulphides, followed by the DP for thiols, and the WB for disulphides. Absolute SC thickness was highest in the body of the GMP (62-80 mum), and generally lowest in the ear (20-38 mum) of all animal types. Relative SC thickness was the same for all animals in the body (40-66%), but lower in the ear (30%). Only -S-S- concentrations were correlated with SC thickness, and primarily in the GMP. Cytochemical analysis showed that high sulphur concentrations were obvious particularly in the CCE of corneal cells in the DP, as compared to the cytoplasm. Intracellular sulphur distribution was homogenous in the WB, and in the GMP, although in the latter at a higher concentration level. The results indicate breed-related effects on keratinisation in porcine corneal cells. Only the SC of the outer side of the ear of DP females is recommended as a model for humans.

The success or failure of the clearance of apoptotic cell remains depends on the ability of phagocytic cells to recognize, phagocytoze, and digest these remains prior to their lysis, which would cause tissue inflammation. We have recently shown that, after mass-induced apoptosis of guinea pig colonocytes in vivo, phagocytosis by resident macrophages, although efficient, does not prevent a pre-inflammatory response of the mucosa. The present study has investigated the cause(s) of this clearance failure. Immunohistochemistry and transmission electron microscopy were applied. Antibodies directed against the epithelial plasma membrane protein E-cadherin, the lysosomal membrane protein LAMP-1, and the lysosomal matrix protease cathepsin-D were used. The results revealed that: (1) anti-E-cadherin labeled the membrane of epithelial apoptotic bodies internalized in macrophages, (2) double and triple labeling demonstrated that the anti-LAMP-1 and anti-cathepsin-D antibodies recognized and were co-localized in lysosomes and/or phagolysosomes in macrophages but left E-cadherin-positive structures unlabeled, (3) the more numerous were the E-cadherin-positive inclusions in macrophages, the smaller was the number of those that stained positive for lysosomal markers. In parallel with electron microscopy, these findings showed that not all apoptotic bodies phagocytozed by macrophages were subsequently digested, suggesting that the phagocytotic ability of these cells was not matched by their digestive capability.

To investigate the influence of overweight and dietary fat supplementation on lipid and insulin glucose metabolism of Shetland ponies, eight Shetland pony geldings were fed a hypercaloric (30 MJ DE/150 kg bwt. and day) fat diet (10% fat as soybean oil) or a carbohydrate control diet for nine months until ponies gained an overweight of 15%. Afterwards oral glucose tolerance tests (oGTT; 5, 6 mmol/kg bwt.) were performed after a 12 hour fast and after a fast which led to an increase of plasma triglyceride concentrations to a threshold of 3 mmol/l (36-65 hrs.). Plasma concentrations of glucose, insulin, triglycerides and non esterified fatty acids (NEFA) were determined for 480 minutes after the glucose load. Ponys having had received the control diet tended to a higher insulin secretion in case of both oGTTs, whereas the glucose tolerance was similar in both groups but lower than in ponies of normal weight. During the oGTTs after fasting leading to the plasma triglyceride threshold, triglyceride concentrations decreased significantly (p < 0.05) faster and stronger in fat fed ponies. Additionally, fat fed pony showed significantly (p < 0.05) lower NEFA levels. The results of this study demonstrate a positive effect of fat feeding on the triglyceride clearance of overweight Shetland ponies.

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.

Absorption of short-chain fatty acids (SCFA) and ammonia implies considerable fluxes of protons across the epithelium of the large intestine. Efficient regulation of intracellular pH (pH(i)) is therefore essential in these cells. The aim of the present study was to examine the effects of SCFA and of ammonia on pH(i), on pH(i) regulation and to characterize the mechanisms involved in pH(i) regulation in surface enterocytes of the guinea-pig caecal and colonic mucosa. Intact epithelia from the caecum and the distal colon were mounted in a microperfusion chamber. pH(i) was measured by fluorescence microscopy using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Addition of SCFA or ammonia to the serosal side changed the enterocyte pH(i) markedly, whereby ammonia caused larger changes in pH(i) than SCFA. In contrast, addition of SCFA to the mucosal solution had no effect on pH(i) and ammonia increased pH(i) only slightly. Basolaterally located pH(i) regulation mechanisms, Na(+)-H(+) exchange and Cl(-)-HCO(3)(-) exchange, are involved mainly in returning pH(i) to normal values. It is concluded that, due to apparently lower permeability of the apical membranes, the caecal and colonic epithelium is protected against pH(i) disturbances caused by the naturally high luminal SCFA and NH(3) concentrations. The major regulation mechanisms of pH(i) are located in the basolateral membrane of the enterocytes.

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.

Short-chain fatty acids (SCFA) in particular butyrate are regarded as an energy source acting in beneficial, protective manner on the colonic mucosa. Previous investigations showed that the colonic mucosa bathed in Ussing chamber with a solution lacking butyrate induced massive apoptosis of epithelial cells. The apoptotic material (bodies and cells) was shed at the mucosa surface. In the present study we aimed to investigate the effects caused in vivo on the colonic mucosa by the absence of butyrate. For this purpose the colon of guinea pigs was perfused in situ with solutions either containing or lacking butyrate. The results show that within 2h of perfusion without butyrate a large amount of epithelial cells underwent apoptosis as in the in vitro experiments. However, apoptotic material instead to be extruded at the epithelial surface accumulates into the intercellular spaces from which it becomes removed by an unusual high number of macrophages. These, engorged with phagocytozed material, lie assembled in a layer below the epithelium. Similar alterations have not been observed after perfusion in the presence of butyrate. The results suggest that this SCFA may protect the colonic mucosa in that it prevents apoptosis. The alterations occurring during 2h of its absence allow to assume that a protracted butyrate deprivation may lead to a breakdown of the integrity of the mucosa thus influencing differently the activity of the macrophages.