Drinking water application of Denagard® Tiamulin for control of Brachyspira pilosicoli infection of laying poultry

Avian intestinal spirochaetosis (AIS) caused by Brachyspira spp., and notably Brachyspira pilosicoli, is common in layer flocks and reportedly of increasing incidence in broilers and broiler breeders. Disease manifests as diarrhoea,increased feed consumption, reduced growth rates and occasional mortality in broilers and these signs are shown in layers also associated with a delayed onset of lay, reduced egg weights, faecal staining of eggshells and non-productive ovaries. Treatment with Denagard® Tiamulin has been used to protect against B. pilosicoli colonisation, persistence and clinical presentation of AIS in commercial layers, but to date there has been no definitive study validating efficacy. Here, we used a poultry model of B. pilosicoli infection of layers to compare the impact of three doses of Denagard® Tiamulin. Four groups of thirty 17 week old commercial pre-lay birds were all challengedwith B. pilosicoli strain B2904with three oral doses two days apart. All birdswere colonised within 2 days after the final oral challenge and mild onset of clinical signs were observed thereafter. A fifth group that was unchallenged and untreated was also included for comparison as healthy birds. Five days after the final oral Brachypira challenge three groups were given Denagard® Tiamulin in drinking water made up following the manufacturer's recommendations with doses verified as 58.7 ppm, 113 ppm and 225 ppm. Weight gain body condition and the level of diarrhoea of birds infected with B. pilosicoli were improved and shedding of the organism reduced significantly (p = 0.001) following treatment with Denagard® Tiamulin irrespective of dose given. The level and duration of colonisation of organs of birds infected with B. pilosicoli was also reduced. Confirming previous findings we showed that the ileum, caeca, colon, and both liver and spleen were colonised and here we demonstrated that treatment with Denagard® Tiamulin resulted in significant reduction in the numbers of Brachyspira found in each of these sites and dramatic reduction in faecal shedding (p b 0.001) to approaching zero as assessed by culture of cloacal swabs. Although the number of eggs produced per bird and the level of eggshell staining appeared unaffected, egg weights of treated birds were greater than those of untreated birds for a period of approximately two weeks following treatment. These data conclusively demonstrate the effectiveness of Denagard® Tiamulin in reducing B. pilosicoli infection in laying hens.

Dystrophin abnormalities in polymyositis and dermatomyositis.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

Characterisation of dystrophin in fetuses at risk for Duchenne muscular dystrophy.

Dystrophin, the product of the Duchenne muscular dystrophy (DMD) gene, was studied in muscle from 16 human fetuses at risk for the disease. Eleven high risk (greater than 95% probability) and 5 low-risk (less than 25% probability) fetuses were studied with antibodies raised to different regions of the protein. All low-risk fetuses showed a similar pattern to that of normal fetuses of a comparable age: using Western blot analysis, a protein was detected of similar size and abundance to that of normal fetuses (i.e. smaller molecular weight than that of adult muscle); immunocytochemistry showed uniform sarcolemmal staining in fetuses older than 18 weeks gestation and differential staining of myotubes at different stages of development (distinguished by size) in younger fetuses (less than 15 weeks gestation). In contrast, Western blot analysis of high-risk fetuses detected low levels of dystrophin in 4 cases; 7 fetuses had no detectable protein. Immunocytochemistry with some dystrophin antibodies showed weak staining of the sarcolemma and around central nuclei in younger fetuses; in older fetuses there was little sarcolemmal staining with any antibody other than occasional positive fibres. These results indicate that careful study of dystrophin in fetuses at risk for DMD can be used to establish the clinical phenotype and provide additional information for future family counselling.

Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

Dystrophin analysis using a panel of anti-dystrophin antibodies in Duchenne and Becker muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in 19 patients with Xp21 disorders and in 25 individuals with non-Xp21 muscular dystrophy. Antibodies raised to seven different regions spanning most of the protein were used for immunocytochemistry. In all patients specific dystrophin staining anomalies were detected and correlated with clinical severity and also gene deletion. In patients with Becker muscular dystrophy (BMD) the anomalies detected ranged from inter- and intra-fibre variation in labelling intensity with the same antibody or several antibodies to general reduction in staining and discontinuous staining. In vitro evidence of abnormal dystrophin breakdown was observed reanalysing the muscle of patients, with BMD and not that of non-Xp21 dystrophies, after it has been stored for several months. A number of patients with DMD showed some staining but this did not represent a diagnostic problem. Based on the data presented, it was concluded that immunocytochemistry is a powerful technique in the prognostic diagnosis of Xp21 muscular dystrophies.

Characterisation of dystrophin during development of human skeletal muscle.

Dystrophin, the 427 x 10(3) Mr product of the Duchenne muscular dystrophy (DMD) gene, was studied in human foetal skeletal muscle from 9 to 26 weeks of gestation. Dystrophin could be detected from at least 9 weeks of gestation at the sarcolemmal membrane of most myotubes, though there was differential staining with antibodies raised to various regions of the protein. Dystrophin immunostaining increased and became more uniform with age and by 26 weeks of gestation there was intense sarcolemmal staining of all myotubes. On a Western blot, a doublet of smaller relative molecular mass than that seen in adult tissue was detected in all foetuses studied. There was a gradual increase in abundance of the upper band from 9 to 26 weeks, and the lower band, although present in low amounts in young foetuses, increased significantly between 20 and 26 weeks of gestation. These data indicate that there are several specific isoforms of dystrophin present in developing skeletal muscle, though the role of these is unknown.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Distribution analysis of the putative cancer marker S100A4 across invasive squamous cell carcinoma penile tissue

MS-based proteomic methods were utilised for the first time in the discovery of novel penile cancer biomarkers. MALDI MS imaging was used to obtain the in situ biomolecular MS profile of squamous cell carcinoma of the penis which was then compared to benign epithelial MS profiles. Spectra from cancerous and benign tissue areas were examined to identify MS peaks that best distinguished normal epithelial cells from invasive squamous epithelial cells, providing crucial evidence to suggest S100A4 to be differentially expressed. Verification by immunohistochemistry resulted in positive staining for S100A4 in a sub-population of invasive but not benign epithelial cells.

Stromal-derived factor-1 alpha (CXCL12) levels increase in periodontal disease

Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions

P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Direct pulp capping with a self-etching adhesive system: Histopathologic evaluation in dogs` teeth

Objective. This study evaluated histopathologically the response of pulp and periradicular tissues after pulp capping with an all-in-one self-etching adhesive system in dogs` teeth. Study design. Forty teeth of 4 dogs were assigned to 3 groups according to the pulp capping material: G1 (n = 20): self-etching adhesive system; G2 (n = 10): Ca(OH)(2); G3 (n = 10): zinc oxide-eugenol. The animals were killed 7 and 70 days after pulp capping. The pieces containing the pulp-capped teeth were removed and processed for histologic analysis. Results. At 7 days, no dentin bridge formation was observed; G1 and G3 exhibited inflammatory pulpal alterations, whereas G2 presented only mild inflammatory infiltrate in the pulp tissue adjacent to the capping material, the remainder being intact. At 70 days, no specimen in G1 or G3 presented dentin bridge formation. The remaining pulp tissue exhibited severe inflammatory alterations and areas of necrosis. In G2, all specimens showed dentin bridge formation and absence of inflammation and mineralized tissue resorption. No bacteria were identified using Brown and Brenn staining techniques in all 3 groups at any observation period. Conclusion. According to the conditions of this study, direct pulp capping with the self-etching adhesive system did not allow pulp tissue repair and failed histopathologically in 100% of the cases. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e34-e40)

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation. Reproduction (2011) 142 593-603

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.