Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Hyperbranched glycopolymers for blood biocompatibility.

Carbohydrate-based drug and gene delivery carriers are becoming extremely popular for in vitro and in vivo applications. These carriers are found to be nontoxic and can play a significant role in targeted delivery. However, the interactions of these carriers with blood cells and plasma components are not well explored. To the best of our knowledge, there are currently no reports that explore the role of carbohydrate based carriers for blood biocompatibility. Hyperbranched glycopolymers of varying molecular weights are synthesized by reversible addition-fragmentation chain transfer polymerization (RAFT) and are studied in detail for their biocompatibility, including hemocompatibility and cytotoxicity against different cell lines in vitro. The hemocompatibility studies (such as hemolysis and platelet activation) indicate that hyperbranched glycopolymers of varying molecular weights produced are highly hemocompatible and do not induce clot formation, red blood cell aggregation, and immune response. Hence, it can be concluded that glycopolymers functionalized carriers can serve as an excellent candidate for various biomedical applications. In addition, cytotoxicity of these hyperbranched polymers is studied in primary and malignant cell lines at varying concentrations using cell viability assay.

In vitro opsonin-independent interactions between cells of smooth and rough phenotypes of Flavobacterium psychrophilum and rainbow trout (Oncorhynchus mykiss) head kidney macrophages.

The cytotoxic activity of smooth and rough phenotypic cells of the fish pathogenic Gram-negative bacterium Flavobacterium psychrophilum to rainbow trout (Oncorhynchus mykiss) head kidney macrophages was investigated in vitro. The cytotoxicity to macrophages was significantly higher for rough cells compared with the smooth cells. The cytotoxic activity increased for both cell types with increasing temperature and the cells retained their cytotoxic nature after metabolical inactivation by heat, suggesting a cell-bound cytotoxic mechanism. The cytotoxicity was significantly reduced in both cell types after treatment with sodium (meta)periodate, indicating that the major bacterial structure involved in the cytotoxicity is of carbohydrate nature. Trypsin treatment further reduced the cytotoxicity in smooth cells, while sialic acid treatment reduced the cytotoxicity in rough cells, suggesting different lysing mechanisms for the two phenotypic variants. The results from the present study therefore suggest that the cytotoxic activity of F. psychrophilum to rainbow trout macrophages in vitro is stronger expressed in the rough phenotype and that it is opsonin-independent and initiated by binding of bacterial surface carbohydrates to lectins on the surface of the macrophages. How the lysis of the macrophages is executed is still unclear but it is suggested to function by different mechanisms in the smooth and the rough cells. The migration of rainbow trout macrophages toward smooth and rough cells of F. psychrophilum was further investigated. The results show that the macrophages were able to recognize both cell types, but the migration rate did not differ between the two phenotypes.

A comprehensive evaluation of the toxicology of cigarette ingredients: carbohydrates and natural products.

CONTEXT: Eleven carbohydrates and natural product ingredients were added individually to experimental cigarettes. OBJECTIVE: A battery of tests was used to compare toxicity of mainstream smoke from these experimental cigarettes to matched control cigarettes without test ingredients. MATERIALS AND METHODS: Smoke fractions from each cigarette type were evaluated using analytical chemistry; in vitro cytotoxicity (neutral red uptake) and in vitro bacterial (Salmonella) mutagenicity (five strains) testing. For 10 ingredients (ß-cyclodextrin, cleargum, D-sorbitol, high fructose corn syrup, honey, invert sugar, maltodextrin, molasses, raisin juice concentrate, and sucrose), 90-day nose-only smoke inhalation studies using rats were also performed. RESULTS: In general, addition of each ingredient in experimental cigarettes resulted in minimal changes in smoke chemistry; the exceptions were D-sorbitol and sucrose, where reductions in amount of 60% to 80% of control values for some smoke constituents were noted. Additionally, each ingredient resulted in small increases in smoke formaldehyde concentrations. Except for a reduction in cytotoxicity by inclusion of maltodextrin and an increase by inclusion of plum juice concentrate, the cytotoxicity and mutagenicity results were unaffected by addition of the other ingredients in experimental cigarettes. There were also very few statistically significant differences within any of the 10 inhalation studies, and when present, the differences were largely sporadic and inconsistent between sexes. CONCLUSION: The carbohydrates and natural products tested here as ingredients in experimental cigarettes as a class increased formaldehyde, but resulted in minimal toxicological responses, even at high inclusion levels compared with the levels used in commercial cigarette products.

Improved osmotic tolerance and ethanol production of ethanologenic Escherichia coli by IrrE, a global regulator of radiation-resistance of Deinococcus radiodurans.

Successful fermentations to produce ethanol using ethanologenic Escherichia coli require tolerance to high concentrations of sugars. Here we demonstrate that irrE, encoding a regulatory protein for radiation-resistance in Deinococcus radiodurans, conferred improved osmotic stress tolerance to E. coli. Expression of the gene protected E. coli cells against 25% glucose or xylose, acid shock. It also markedly improved cellular viability, the transcriptional levels of trehalose biosynthetic genes (otsBA) and trehalose content in the IrrE-expressing strain compared with the control strain. IrrE expression also enhanced the expression levels and enzymatic activities of PDC and ADHB as well as ethanol production. Our results suggest that IrrE could potentially be used to improve osmotic stress tolerance and ethanol production in ethanologenic strains.

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis.

REASONS FOR PERFORMING STUDY: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model. OBJECTIVES: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. METHODS: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV group, n=8) or at onset of Obel grade 1 lameness (OG1 group, n=8). Control horses (CON group, n=8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time-quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. RESULTS: Increased mRNA concentrations (P<0.05) for IL-1ß, IL-6, IL-12p35, COX-2, E-selectin and ICAM-1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL-2, IL-4, IL-10, TNF-α, IFN-γ or COX-1 at either the DEV or OG1 time points. CONCLUSIONS: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. POTENTIAL RELEVANCE: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.

Cryopreservation of scalps of Malaysian bananas using a pre-growth method.

The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 square cm) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light and 8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40 degree C for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg per liter benzylaminopurine, 0.2 mg per liter indole acetic acid and 10 mg per liter ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48 percent of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10 percent, 13 percent, 42 percent and 48 percent for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.

Citrin deficiency and current treatment concepts.

In this paper, we describe the historical aspects of citrin and citrin deficiency, characteristic food preference and food aversion of citrin-deficient subjects, and carbohydrate toxicity in relation to ureogenesis and issues of the conventional treatment procedures for hyperammonemia in citrin deficiency, leading to current treatment concepts for citrin deficiency. We also emphasize the importance of a citrin deficiency mouse model in elucidating the pathophysiology and developing novel therapeutics based on the pathophysiology, such as sodium pyruvate.

Field experiments of Anopheles gambiae attraction to local fruits/seedpods and flowering plants in Mali to optimize strategies for malaria vector control in Africa using attractive toxic sugar bait methods.

BACKGROUND: Based on recent studies in Israel demonstrating that attractive toxic sugar bait (ATSB) methods can be used to decimate local anopheline and culicine mosquito populations, an important consideration is whether the same methods can be adapted and improved to attract and kill malaria vectors in Africa. The ATSB approach uses fruit or flower scent as an attractant, sugar solution as a feeding stimulant, and an oral toxin. The ATSB solutions are either sprayed on vegetation or suspended in simple bait stations, and the mosquitoes ingesting the toxic solutions are killed. As such, this approach targets sugar-feeding female and male mosquitoes. This study examines the attractiveness of African malaria vectors to local fruits/seedpods and flowering plants, key biological elements of the ATSB approach for mosquito control. METHODS: Three field experiments were conducted at sites in Mali. The attraction of Anopheles gambiae s.l. to 26 different local fruits and seedpods was determined at a site in the semi-arid Bandiagara District of Mali. Wire mesh glue traps with fruits/seedpods suspended on skewers inside were set along a seasonal lagoon. Seven replicates of each fruit/seedpod species were tested, with a water-soaked sponge and a sugar-soaked sponge as controls. The attraction of An. gambiae s.l. to 26 different types of flowering plants was determined at a site near Mopti in Mali. The flowering plants held in a water-filled buried container were tested using the same glue traps, with controls including water only and sugar solution. Six replicates of each selected plant type were tested on transects between rice paddies. Additional studies using CDC light traps were done to determine the relative densities and periodicity of An. gambiae s.l. attraction to branches of the most highly attractive flowering plant, branches without flowers, human odor, and candescent light. RESULTS: Of the 26 fruits and seedpods tested, 6 were attractive to An. gambiae s.l. females and males, respectively. Guava (Psidium guajava) and honey melon (Cucumis melo) were the two most attractive fruits for both females and males. Of the 26 flowering plants tested, 9 were significantly attractive for females, and 8 were attractive for males. Acacia macrostachya was the most attractive flowering plant. Periodicity studies using this plant showed peaks of An. gambiae s.l. attraction between 1930 and 2200 h and 0400-0500 h, which differed considerably from the response to human odors, which expectedly peaked at around midnight. CONCLUSION: These field experiments in Mali highlight that female and male An. gambiae s.l. have pronounced differences in attraction for diverse types of indigenous fruits/seedpods and flowering plants. The identification of attractive fruits and seedpods shows that a variety of indigenous and locally abundant natural products could potentially be used as juices to make ATSB solution for mosquito control. As well, the simple methods used to identify the most attractive flowering plants provide valuable insights into the natural history of sugar feeding for An. gambiae s.l. These observations can be used to guide future strategies for employing ATSB methods for malaria vector control in Africa. They also provide a basis for subsequent chemical analysis and development of attractive baits for mosquito control.

Chemical structure and mechanism of polysaccharide on Pb2+ tolerance of Cordyceps militaris after Pb2+ domestication.

In this study, the purified polysaccharide (DCP-I) was extracted from Cordyceps militaris domesticated with Pb2+. After that, the structural feature and mechanism of lead resistance of DCP-I were investigated using novel approaches. The results showed that the average molecular weight of DCP-I was 1.206 × 103 kDa and mainly consist of Rhamnose, Galactose, Glucose, Galacturonic acid and Glucuronic acid in a molar ratio of 0.130:47.687:40.784:1.795:0.48. Besides, the main chain of DCP-I was composed by →6)-Galp-(1→, →4)-Glcp-(1→ and →1,4)-Glcp-(6→, while the side chain was →1)-Rhaf-(2→ and D-Glcp-(1→, and the DCP-I contained Alacturonic acid and Glucuronic acid. In addition, the result of Congo red test showed that DCP-I did not exist triple-helical structures. SEM, EDX and XPS analyses results showed that the functional groups of DCP-I related to C, H and O (-OH, -COOH and -C=O) could combined with Pb2+effectively. The adsorption processes were described by the Pseudo-second-order kinetic model (R2 = 0.9978) and Langmuir isotherm (R2 = 0.9979) for Pb2+ indicating that adsorption process of DCP-I to Pb2+ was a kind of single molecular layer chemical adsorption.

Leukocyte-derived and endogenous matrix metalloproteinases in the lamellae of horses with naturally acquired and experimentally induced laminitis.

RATIONALE: Inflammation and dysregulation of endogenous matrix metalloproteinase (MMP) production are implicated in the development of equine laminitis. In this study, we examine quantitative relationships among levels of leukocyte-derived proMMP-9 and MMP-9, lamellar proMMP-2 and MMP-2, and expression of proMMP-2 processing enzymes, MT1-MMP/PACE4, as steps towards determining whether inflammation and dysregulation of endogenous MMP production are independent or co-dependent processes. ANIMALS: Archived samples of lamellae from horses with naturally acquired laminitis (n = 12), and from horses administered a pro-laminitic gastric bolus of starch gruel were used, the latter horses falling into two groups: (i) responders (CHO-R, n = 7), which developed Obel grade 3-lameness and (ii) non-responders (CHO-NR, n = 4), which did not become lame. METHODS: Lamellar tissue extracts were analyzed by gelatin zymography to determine gelatinase content and by a myeloperoxidase ELISA to quantify relative monocyte/neutrophil content in the tissue. Real-time PCR was employed to measure gene expression of MT1-MMP and PACE4. RESULTS AND CONCLUSIONS: Extracts of lamellae from control horses, CHO-NR and horses with chronic (non-aggravated) laminitis had similarly low levels of pro and processed MMP-9 and MMP-2. In contrast, proMMP-9 was significantly elevated in extracts of lamellae from CHO-R and horses with naturally acquired acute and aggravated chronic laminitis. Lamellar MMP-2 was also increased significantly in the CHO-R and aggravated chronic laminitis groups, although not in the horses with naturally acquired acute laminitis. Concentrations of proMMP-9 correlated directly with myeloperoxidase content in lamellar extracts, suggesting production/induction by inflammatory leukocytes. In contrast, concentrations of proMMP-2 and MMP-2 were unrelated to concentrations of myeloperoxidase or proMMP-9 suggesting that leukocyte infiltration and dysregulation of endogenous MMP-2 are independent processes most likely with distinct inducers. Neither MT1-MMP nor PACE4 gene expression was elevated relative to controls in any group; this is discussed with respect to proMMP-2 processing in disease. In addition, variability in relative concentrations of lamellar MMPs observed among horses with Obel grade 3-lameness is discussed in the context of laminitis risk assessment and disease outcome.

Cell response to magnetic glyconanoparticles: does the carbohydrate matter?

Magnetic nanoparticles have been used in therapeutic and diagnostic approaches in biomedicine for many years. For these applications, it is very important to investigate the nanoparticle-cell interactions. In this study we report a simple method for the preparation of gold-iron nanoparticles protected and functionalized with biologically relevant saccharides (maltose, lactose, and glucose). The nanoparticles were subsequently tested in vitro with a human fibroblast cell line to determine biocompatibility, and the cell-particle interactions, using fluorescence and scanning electron microscopies. Different cellular responses were obtained for each type of glyconanoparticle, demonstrating that the cells can recognize the saccharides on the nanoparticles.

Sialoglycoprotein and carbohydrate complexes in chromium toxicity.

Chromium(VI) compounds are amongst the most widely encountered industrial carcinogens and are of increasing concern with respect to environmental exposure. Sialoglycoproteins and carbohydrates play a crucial role in stabilizing oxoCr(V) intermediates, which are produced by extracellular and intracellular reduction of chromium(VI). Recent research has addressed the molecular characterization of oxoCr(V)-sialoglycoprotein and -carbohydrate complexes and the roles that these species may play in Cr(VI) metabolism and carcinogenesis. Particular highlights include the role of oxoCr(V) complexes of extracellular sialoglycoproteins, intracellular D-glucose, and related species and their potential roles in Cr(VI)-induced genotoxicity.

V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals.

We recently constructed a Chinese hamster V79-derived cell line that stably expresses human cytochrome P450 (CYP) 2E1 and human sulphotransferase (SULT) 1A1. These enzymes are involved in the bioactivation of numerous promutagens/procarcinogens, but are not taken into account in standard in vitro mutagenicity assays. Various carbohydrate pyrolysis products and other food contaminants that induce tumours or preneoplastic lesions in laboratory animals are inactive or only weakly active in standard in vitro genotoxicity assays. This is the case for acrylamide, furan, 5-hydroxymethylfurfural, nitrofen and N-nitrosodimethylamine. These compounds were investigated for induction of sister chromatid exchange (SCE) in V79-hCYP2E1-hSULT1A1 cells. All test compounds showed positive results over a wide concentration range, starting at 0.01 microM for N-nitrosodimethylamine, 3 microM for furan, 12.5 microM for nitrofen, 20 microM for 5-hydroxymethylfurfural, and 200 microM for acrylamide. The concentration-response curve of furan was unusual, as this compound induced a statistically significant, but rather constant and weak increase in SCE over an extremely wide concentration range (3-16,000 microM). Furan was slightly less active, whereas the remaining compounds were much less active in the parental V79 cell line than in V79-hCYP2E1-hSULT1A1 cells. Compared to many other genotoxic effects, the study of SCE only requires small numbers of cells (and incubation volumes) and usually is detected even at low concentrations of the genotoxicant. Therefore, induction of SCE in V79-hCYP2E1-hSULT1A1 cells may be useful in the genotoxicity testing of preparations of heated food and in their bioassay-directed fractionation.

Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation.

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.

Control of sand flies with attractive toxic sugar baits (ATSB) and potential impact on non-target organisms in Morocco.

BACKGROUND: The persistence and geographical expansion of leishmaniasis is a major public health problem that requires the development of effective integrated vector management strategies for sand fly control. Moreover, these strategies must be economically and environmentally sustainable approaches that can be modified based on the current knowledge of sand fly vector behavior. The efficacy of using attractive toxic sugar baits (ATSB) for sand fly control and the potential impacts of ATSB on non-target organisms in Morocco was investigated. METHODS: Sand fly field experiments were conducted in an agricultural area along the flood plain of the Ourika River. Six study sites (600 m x 600 m); three with "sugar rich" (with cactus hedges bearing countless ripe fruits) environments and three with "sugar poor" (green vegetation only suitable for plant tissue feeding) environments were selected to evaluate ATSB, containing the toxin, dinotefuran. ATSB applications were made either with bait stations or sprayed on non-flowering vegetation. Control sites were established in both sugar rich and sugar poor environments. Field studies evaluating feeding on vegetation treated with attractive (non-toxic) sugar baits (ASB) by non-target arthropods were conducted at both sites with red stained ASB applied to non-flowering vegetation, flowering vegetation, or on bait stations. RESULTS: At both the sites, a single application of ATSB either applied to vegetation or bait stations significantly reduced densities of both female and male sand flies (Phlebotomus papatasi and P. sergenti) for the five-week trial period. Sand fly populations were reduced by 82.8% and 76.9% at sugar poor sites having ATSB applied to vegetation or presented as a bait station, respectively and by 78.7% and 83.2%, respectively at sugar rich sites. The potential impact of ATSB on non-targets, if applied on green non-flowering vegetation and bait stations, was low for all non-target groups as only 1% and 0.7% were stained with non-toxic bait respectively when monitored after 24 hours. CONCLUSIONS: The results of this field study demonstrate ATSB effectively controls both female and male sand flies regardless of competing sugar sources. Furthermore, ATSB applied to foliar vegetation and on bait stations has low non-target impact.

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

REASONS FOR PERFORMING STUDY: Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES: To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN: Experimental study. METHODS: Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS: Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS: Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Laminar inflammatory events in lean and obese ponies subjected to high carbohydrate feeding: Implications for pasture-associated laminitis.

REASONS FOR PERFORMING STUDY: Acute, massive enteral carbohydrate overload is associated with laminar inflammation in equids; it is unclear if the same is true for a more prolonged period of moderate dietary carbohydrate intake. OBJECTIVES: To characterise laminar inflammation in ponies exposed to a dietary carbohydrate challenge meant to mimic acute pasture exposure. STUDY DESIGN: In vivo experiment. METHODS: Mixed-breed ponies (n = 22) received a diet of hay chop (nonstructural carbohydrate [NSC] ∼7% on a dry matter [DM] basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following dietary acclimation, ponies were stratified into either Lean (n = 11, body condition score [BCS] ≤4) or Obese (n = 11, BCS ≥7) groups and each group further stratified to either remain on the control, low NSC diet (n = 5 each for Obese and Lean) or receive a high NSC diet (hay chop supplemented with sweet feed and oligofructose, total diet ∼42% NSC; n = 6 each for Obese and Lean) for a period of 7 days. Laminar samples were collected following euthanasia and sections stained immunohistochemically for CD163, MAC387/calprotectin and cyclo-oxygenase-2 (COX-2) using commercially available antibodies. The number of CD163 (+) and MAC387(+) cells was quantified for each section; the distribution of COX-2 expression was qualitatively assessed. Laminar mRNA concentrations of several proinflammatory molecules (interleukin-1ß [IL-1ß], IL-6, tumour necrosis factor-α [TNFα], IL-8, IL-10, monocyte chemoattractant protein-1 [MCP-1], MCP-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, plasminogen activator inhibitor-1 (PAI-1) and COX-2 were evaluated using real-time quantitative polymerase chain reaction (qPCR). RESULTS: High carbohydrate feeding resulted in no increase in laminar proinflammatory cytokine expression; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. CONCLUSIONS: These results suggest that the marked laminar inflammation observed in models of sepsis-associated laminitis may not play a central role in the pathophysiology of pasture-associated laminitis.

Bacterial endotoxin (lipopolysaccharide) stimulates the rate of iron oxidation.

Bacterial endotoxin (lipopolysaccharide) has affinity for a number of cations, including iron. Previous investigations have demonstrated that lipopolysaccharide can affect the oxidation rate of iron; heme-bound ferrous iron in hemoglobin is oxidized to ferric iron when hemoglobin binds lipopolysaccharide. In the present study, we directly examined the interaction between lipopolysaccharide and iron. Lipopolysaccharide caused a concentration-dependent increase in the rate of iron oxidation, with up to a 23-fold increase in oxidation in the presence of 200 microg/ml Escherichia coli lipopolysaccharide. This effect was seen both with several carbohydrate-rich smooth lipopolysaccharides and also with carbohydrate-poor rough lipopolysaccharide. Extensively deacylated rough lipopolysaccharide had no effect, suggesting a role of the fatty acid components of lipopolysaccharide in this process. Purified lipid A produced inconsistent results: some preparations stimulated iron oxidation and others did not. A series of sugars, starches and a preparation of purified O-chain polysaccharide (the carbohydrate portion of the lipopolysaccharide macro-molecule) had no effect on the rate of iron oxidation, whereas phospholipid-enriched brain tissue extracts (similar to the lipid A component of lipopolysaccharide) stimulated oxidation. We conclude that the lipid moiety of bacterial lipopolysaccharide is responsible for the stimulation of iron oxidation. This process may contribute to the ability of lipopolysaccharide to cause oxidation of heme-bound iron in hemoglobin.

Imino sugars that are less toxic but more potent as antivirals, in vitro, compared with N-n-nonyl DNJ.

Imino sugar glucosidase inhibitors have selective antiviral activity against certain enveloped, mammalian viruses. Deoxynojirimycins (DNJs) modified by N-alkylation to contain a nine carbon atom side chain (N-n-nonyl-deoxynojirimycin; N-nonyl-DNJ, NN-DNJ) were shown to be, for example, at least 20 times more potent in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays than the non-alkylated DNJ. These data suggested that modification of the alkyl side chain could influence antiviral activity. Previous work has focused on varying side chain length. In this report, the influence of side chain branching and cyclization upon toxicity and antiviral activity was explored. Briefly, using a virus secretion assay for HBV and a single step growth (yield reduction) assay for BVDV, 14 different DNJ-based sugars, possessing various N-alkyl substitutions, were tested for antiviral activity. Of the series, N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ were determined to have the best selectivity index against BVDV and HBV, with the N-methoxy analogue being the most potent with micromolar antiviral activity. The results of this antiviral survey and the implications for the mechanism of action and ultimate therapeutic potential of the DNJ-based imino sugars is provided and discussed.