Evaluation of a zinc chelate on clinical swine dysentery under field conditions.

BACKGROUND: Brachyspira hyodysenteriae is the primary cause of swine dysentery, characterized by bloody to mucoid diarrhea due to mucohaemorhagic colitis in pigs and primarily affects pigs during the grow/finishing stage. Control and prevention of B. hyodysenteriae consists of administration of antimicrobial drugs, besides management and adapted feeding strategies. A worldwide re-emergence of the disease has recently been reported with an increasing number of isolates demonstrating decreased susceptibility to several crucially important antimicrobials in the control of swine dysentery. A novel non-antibiotic zinc chelate has been reported to demonstrate positive effects on fecal quality and consistency, general clinical signs, average daily weight gain and B. hyodysenteriae excretion during and after a 6-day oral treatment. The objective of the present study was to evaluate the zinc chelate (Intra Dysovinol® 499 mg/ml (ID); Elanco) on naturally occurring swine dysentery due to B. hyodysenteriae under field conditions in the Netherlands. RESULTS: Oral administration of zinc chelate resulted in improvement of general clinical signs from 3 days onwards in the ID-treated group combined with a significantly better total fecal score at 14 days post-treatment. Overall, average daily weight gain was better in the ID-treated group over the entire study period (0-14 days) and during the 8 days following the end of ID-treatment. A significant reduction (4.48 vs. 0.63 log10 cfu/g feces; ID-treated vs. control) in B. hyodysenteriae excretion was observed during the 6-day treatment period with a high percentage of animals (58.3 vs. 12.3%; ID-treated vs. control) with no excretion of B. hyodysenteriae from their feces. No additional antimicrobial treatment was needed in the ID-treated group, whereas 35% of the pigs in the control group were treated with an antibiotic at least once. No mortality occurred in both groups. No adverse events were reported during and following the ID-treatment. CONCLUSIONS: Zinc chelate - administered as a Zn-Na2-EDTA complex - is a non-antibiotic treatment for swine dysentery that reduces B. hyodysenteriae shedding with 4.48 log10 cfu/g feces within its 6-day treatment while improving general clinical signs (90.0 vs. 73.6% animals with normal score) and total fecal score within 2-4 days following administration in naturally infected pigs. The positive effects of ID treatment remain for at least 8 days after cessation of oral ID therapy. Pigs remaining in a highly contaminated environment may be re-infected following the end of ID treatment, however, this is not different to standard antimicrobial therapy. Therefore, control of swine dysentery should combine an efficacious treatment with additional management practices to reduce the environmental infection pressure in order to limit re-infection as much as possible. The ID treatment resulted in a higher growth rate and improved general health, whereas no mortality was observed and no additional therapeutic treatments were necessary in contrast to the control pigs.

Treatment of clinical Brachyspira hyodysenteriae with zinc chelate in pigs: a blinded, randomised controlled trial.

BACKGROUND: Brachyspira hyodysenteriae infection in pigs ('swine dysentery') leads to decreased feed conversion, growth losses and mortality. Current countermeasures have the downside of antibiotic resistance (antibiotics) and ecotoxicity (zinc oxide). The aim of this study was to evaluate the effect of a novel zinc chelate (Intra Dysovinol (ID)) on clinical signs of swine dysentery and shedding of B hyodysenteriae under field conditions. METHODS: In a randomised, double-blinded, controlled trial under Good Clinical Practice on two commercial farms, 58 B hyodysenteriae positive pigs from 16 pens received drinking water containing ID, or placebo, during six consecutive days. Faecal quality (consistency, colour, additions) was scored and faeces were analysed for presence of B hyodysenteriae by PCR. ID treatment positively affected faecal quality (consistency) and daily growth rates. RESULTS: At the last treatment day, B hyodysenteriae was not detectable in the faeces of any of the ID-treated animals, while all placebo animals remained B hyodysenteriae positive by PCR. All ID-treated animals recovered, while 5 placebo-treated animals died and 12 placebo pigs required additional treatment before the end of the study (up to 14 days after treatment start). CONCLUSION: This non-antibiotic treatment stopped the clinical signs and shedding of B hyodysenteriae in naturally infected pigs.

Breath acetone to monitor life style interventions in field conditions: an exploratory study.

OBJECTIVE: To assess whether breath acetone concentration can be used to monitor the effects of a prolonged physical activity on whole body lipolysis and hepatic ketogenesis in field conditions. METHODS: Twenty-three non-diabetic, 11 type 1 diabetic, and 17 type 2 diabetic subjects provided breath and blood samples for this study. Samples were collected during the International Four Days Marches, in the Netherlands. For each participant, breath acetone concentration was measured using proton transfer reaction ion trap mass spectrometry, before and after a 30-50 km walk on four consecutive days. Blood non-esterified free fatty acid (NEFA), beta-hydroxybutyrate (BOHB), and glucose concentrations were measured after walking. RESULTS: Breath acetone concentration was significantly higher after than before walking, and was positively correlated with blood NEFA and BOHB concentrations. The effect of walking on breath acetone concentration was repeatedly observed on all four consecutive days. Breath acetone concentrations were higher in type 1 diabetic subjects and lower in type 2 diabetic subjects than in control subjects. CONCLUSIONS: Breath acetone can be used to monitor hepatic ketogenesis during walking under field conditions. It may, therefore, provide real-time information on fat burning, which may be of use for monitoring the lifestyle interventions.

Upregulation of skeletal muscle inflammatory genes links inflammation with insulin resistance in women with the metabolic syndrome.

The metabolic syndrome, a combination of interrelated metabolic risk factors, is associated with insulin resistance and promotes the development of cardiovascular diseases and type 2 diabetes mellitus. There is a close link between inflammation and metabolic disease, but the responsible mechanisms remain elusive. The aim of this study was to identify differentially expressed genes in insulin-resistant skeletal muscle tissue of women with the metabolic syndrome compared with healthy control women. Women with the metabolic syndrome (n = 19) and healthy control women (n = 20) were extensively phenotyped, insulin sensitivity was measured using a hyperinsulinaemic euglycaemic clamp, and a skeletal muscle biopsy was obtained. Gene expression levels were compared between the two groups by microarrays. The upregulated genes in skeletal muscle of the women with the metabolic syndrome were primarily enriched for inflammatory response-associated genes. The three most significantly upregulated of this group, interleukin 6 receptor (IL6R), histone deacetylase 9 (HDAC9) and CD97 molecule (CD97), were significantly correlated with insulin resistance. Taken together, these findings suggest an important role for a number of inflammatory-related genes in the development of skeletal muscle insulin resistance.

Repair of surgically created diaphragmatic defect in rat with use of a crosslinked porous collagen scaffold.

Large defects in congenital diaphragmatic hernia are closed by patch repair, which is associated with a high complication risk and reherniation rate. New treatment modalities are warranted. We evaluated the feasibility of using an acellular biodegradable collagen bioscaffold for a regenerative medicine approach to close a surgically created diaphragmatic defect in a rat model. Scaffold degradation, cellular ingrowth and regeneration of the diaphragm were studied. In 25 rats, a subcostal incision was made and one third of the right hemidiaphragm was resected. Crosslinked porous type I collagen scaffolds (Ø ~ 14 mm) were sutured into the lesion. Rats were sacrificed at 2, 4, 8, 12 or 24 weeks after scaffold implantation. Implants were evaluated macroscopically and (immuno)histologically. Survival after surgery was 88% with no evidence of reherniation. Histological examination showed that the collagen scaffold degraded slowly and new collagen, elastin and mesothelium were deposited. Blood vessels were observed primarily at the outer borders of the scaffold; their number gradually increased in time. Muscle fibres were found on the scaffold covering up to 10% of the defect. Macroscopically, adhesion of the scaffold to the liver was observed. Use of a collagen scaffold to close a surgically created diaphragmatic defect is feasible, with evidence of new tissue formation. The use of crosslinked collagen scaffolds allows targeted modification; e.g. addition of growth factors to further stimulate growth of muscle cells.

The identification of genetic pathways involved in vascular adaptations after physical deconditioning versus exercise training in humans.

Physical inactivity and exercise training result in opposite adaptations of vascular structure. However, the molecular mechanisms behind these adaptations are not completely understood. We used a unique study design to examine both vascular characteristics of the superficial femoral artery (using ultrasound) and gene expression levels (from a muscle biopsy) in human models for physical deconditioning and exercise training. Initially, we compared able-bodied control subjects (n = 6) with spinal cord-injured individuals (n = 8) to assess the effects of long-term deconditioning. Subsequently, able-bodied control subjects underwent short-term lower limb deconditioning using 3 weeks of unilateral limb suspension. Spinal cord-injured individuals were examined before and after 6 weeks of functional electrical stimulation exercise training. Baseline femoral artery diameter and hyperaemic flow were lower after short- and long-term deconditioning and higher after exercise training, whilst intima-media thickness/lumen ratio was increased with short- and long-term deconditioning and decreased with exercise training. Regarding gene expression levels of vasculature-related genes, we found that groups of genes including the vascular endothelial growth factor pathway, transforming growth factor ß1 and extracellular matrix proteins were strongly associated with vascular adaptations in humans. This approach resulted in the identification of important genes that may be involved in vascular adaptations after physical deconditioning and exercise.

Expression of genes involved in fatty acid transport and insulin signaling is altered by physical inactivity and exercise training in human skeletal muscle.

Physical deconditioning is associated with the development of chronic diseases, including type 2 diabetes and cardiovascular disease. Exercise training effectively counteracts these developments, but the underlying mechanisms are largely unknown. To gain more insight into these mechanisms, muscular gene expression levels were assessed after physical deconditioning and after exercise training of the lower limbs in humans by use of gene expression microarrays. To exclude systemic effects, we used human models for local physical inactivity (3 wk of unilateral limb suspension) and for local exercise training (6 wk of functional electrical stimulation exercise of the extremely deconditioned legs of individuals with a spinal cord injury). The most interesting subset of genes, those downregulated after deconditioning as well as upregulated after exercise training, contained 18 genes related to both the "insulin action" and "adipocytokine signaling" pathway. Of these genes, the three with strongest up/downregulation were the muscular fatty acid-binding protein-3 (FABP3), the fatty acid oxidizing enzyme hydroxyacyl-CoA dehydrogenase (HADH), and the mitochondrial fatty acid transporter solute carrier 25 family member A20 (SLC25A20). The expression levels of these genes were confirmed using RT-qPCR. The results of the present study indicate an important role for a decreased transport and metabolism of fatty acids, which provides a link between physical activity levels and insulin signaling.

Construction of a microstructured collagen membrane mimicking the papillary dermis architecture and guiding keratinocyte morphology and gene expression.

A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar-structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration.

A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity.

Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials.

An overview of methods for the in vivo evaluation of tissue-engineered skin constructs.

Cutaneous wounding often leads to contraction and scarring, which may result in a range of functional, cosmetic, and psychological complications. Tissue-engineered skin substitutes are being developed to enhance restoration of the skin and improve the quality of wound healing. The aim of this review is to provide researchers in the field of tissue engineering an overview of the methods that are currently used to clinically evaluate skin wound healing, and methods that are used to evaluate tissue-engineered constructs in animal models. Clinically, the quality of wound healing is assessed by noninvasive subjective scar assessment scales and objective techniques to measure individual scar features. Alternatively, invasive technologies are used. In animal models, most tissue-engineered skin constructs studied are at least evaluated macroscopically and by using conventional histology (hematoxylin-eosin staining). Planimetry and immunohistochemistry are also often applied. An overview of antibodies used is provided. In addition, some studies used methods to assess gene expression levels and mRNA location, transillumination for blood vessel observation, in situ/in vivo imaging, electron microscopy, mechanical strength assessment, and microbiological sampling. A more systematic evaluation of tissue-engineered skin constructs in animal models is recommended to enhance the comparison of different constructs, thereby accelerating the trajectory to application in human patients. This would be further enhanced by the embracement of more clinically relevant objective evaluation methods. In addition, fundamental knowledge on construct-mediated wound healing may be increased by new developments in, for example, gene expression analysis and noninvasive imaging.

High density gene expression microarrays and gene ontology analysis for identifying processes in implanted tissue engineering constructs.

The in vivo performance of tissue-engineered constructs is often based on generally accepted read-out parameters, like (immuno)histology. In this study, high-density gene expression microarrays and gene ontology (GO) analysis were used as a read-out tool to identify the biological processes occurring after implantation of an acellular collagen-based skin construct using a rat full-thickness wound model. A freely-available program (DAVID) was used to identify up/downregulated biological processes (GO-terms) and results were compared to wound healing/regeneration without a construct. The entire process from RNA isolation to biological interpretation is explained step-by-step. Conventional (immuno)histology was used to validate the biological processes identified and indicate that microarray analysis may provide a valuable, fast and unbiased tool to evaluate the in vivo performance of tissue-engineered constructs. However, challenges remain e.g. with regards to the development of specific GO-terms and annotation of the (rat) genome.

Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164).

Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation ( approximately 124mg/L) and purified by heparin affinity chromatography. SDS-PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences.