Parturition affects test-positivity in sheep with subclinical paratuberculosis; investigation following a preliminary analysis

Within the context of an investigation focused at improving the effectiveness of test-and-removal for the control of ovine paratuberculosis, we recently conducted a preliminary study using ELISA and real time PCR to assess whether parturition affects test-positivity, in animals with subclinical paratuberculosis. Samples of faeces and blood were collected from 42 adult female animals, before (PP1) and after parturition (PP2), and before mating (PP3). In the preliminary stage of the analysis, only one of the animals tested reacted positively to ELISA (2.38%, 1 of 42), which corresponds to 2.8% of PCR-reactors (1 of 36). Therefore, the final stage the investigation was conducted using only real time PCR, which was applied to test samples of faeces collected from 85 animals, in 5 periods of sampling: 4–15/1–3 days (FP1/FP2) before and after parturition (FP3/FP4), and before mating (FP5). The result of the preliminary analysis indicated that PCR-positivity in terms of the number of shedders and the amount of MAP, is statistically significantly lower before parturition (PP1), whereas that of the final, higher in FP4 compared to FP5. Significantly higher levels of positivity in PP2 and FP4 were also recorded in connection with animals reacting positively to PCR more than once. In conclusion, in sheep with subclinical paratuberculosis, the period of 4–15 days postpartum is more suitable for the application of test-and-removal aiming to the control of the disease, using real time PCR. The use of ELISA for the same purpose is not recommended in the specific category of animals, due to low sensitivity. © 201

Functional analysis of 3'UTR polymorphisms in the caprine SLC11A1 gene and its association with the Mycobacterium avium subsp. paratuberculosis infection

The study aimed to investigate whether the genetic polymorphisms in the 3. 'UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3. 'end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3. 'UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level. © 2015 Elsevier B.V

Cytokine gene therapy in experimental allergic encephalomyelitis by injection of plasmid DNA-cationic liposome complex into the central nervous system.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system with many similarities to multiple sclerosis. The main effector cells involved are CD4+ T cells, recognizing encephalitogenic epitopes within the central nervous system, and macrophages, both of which secrete proinflammatory cytokines, such as IFN-gamma and TNF. Studies have shown that immunomodulation of this inflammatory response by anti-inflammatory cytokines (IL-4, IL-10, IFN-beta, and TGF-beta) can reduce clinical severity in EAE. The importance of TNF in EAE has been demonstrated by using soluble TNF-receptor molecules to inhibit EAE. However, the limitation of this type of therapy is the necessity for frequent administration of cytokine proteins due to their short biologic half-life. This study demonstrates that EAE can be inhibited by a single injection of therapeutic cytokine (IL-4, IFN-beta, and TGF-beta) DNA-cationic liposome complex directly into the central nervous system. DNA coding for a novel, dimeric form of human p75 TNF receptor also ameliorated clinical EAE. Local administration of DNA-cationic liposome complex has identified gene targets that may be more efficiently exploited using vectors producing more stable expression for effective treatment of neuroimmunologic disease

In vitro target validation and in vivo efficacy of p38 MAP kinase inhibition in established chronic collagen-induced arthritis model: a pre-clinical study.

OBJECTIVES: The aim of the present study was to determine the in vivo efficacy of p38 mitogen-activated protein kinase (MAPK) inhibitors, namely GW856553X and GSK678361, in murine models of arthritis. METHODS: The effect of p38 MAPK inhibitors was tested in 2 variants of the collagen-induced arthritis model (CIA) in DBA/1 mice, acute arthritis induced by heterologous collagen and chronic relapsing arthritis induced by homologous collagen. Animals were treated after onset of arthritis. Furthermore, post-onset disease efficacy of GSK678361 was tested in the chronic model, so as to determine the effects on established arthritis. In vitro studies were carried out with GW856553X, using human umbilical vein endothelial cells, to determine potential effects of GW856553X on the vasculature. RESULTS: In both acute and chronic arthritis, GW856553X reduced signs and symptoms of disease, and protected joints from damage. The effect of GW856553X in chronic CIA was confirmed using an alternative compound, GSK678361. Importantly, treatment with GSK678361 from 14 days post-onset of chronic arthritis completely reversed signs of established disease and joint destruction. Mechanism of action studies demonstrated that GW856553X inhibited endothelial cell migration and angiogenesis in vitro, with reduced pro-inflammatory cytokine production. CONCLUSIONS: Suppression of murine CIA by the p38 MAPK inhibitors GW856553X and GSK678361 suggests that they may have therapeutic potential for future use in RA if safe clinical dosing achieves adequate compound exposure

Inhibition of p38 MAPK-dependent bronchial contraction after ozone by corticosteroids

We determined the role of p38 mitogen-activated protein kinase (MAPK) in the increased airway smooth muscle (ASM) contractile responses following ozone and modulation by corticosteroids. Mice were exposed to air or ozone (3 ppm for 3 h) and isometric contractile responses of bronchial rings to acetylcholine (ACh) were measured using a myograph in the presence of p38 MAPK inhibitor, SB239063 (10(−6) M) or dexamethasone (10(−6) M). Because MAPK phosphatase (MKP)-1 is a negative regulator of p38 MAPK, we also studied these effects in MKP-1(-/-) mice. Bronchial rings from ozone-exposed wild-type and MKP-1(-/-) mice showed increased contractile responses, with a leftward shift of the dose–response curve in MKP-1(-/-) mice. SB239063 inhibited bronchial contraction equally in air- and ozone-exposed C57/BL6 and MKP-1(-/-) mice. Dexamethasone inhibited ACh-induced bronchial contraction in both air- and ozone-exposed C57/BL6 mice, but not in air- or ozone-exposed MKP-1(-/-) mice. ACh-stimulated p38 MAPK and heat shock protein (HSP)27 phosphorylation, as measured by Western blotting, and this effect was suppressed by SB239063 in C57/BL6 and MKP-1(-/-) mice, but not by dexamethasone in either air- or ozone-exposed MKP-1(-/-) mice. p38 MAPK plays a role in maximal ACh-induced isometric contractile responses and increased contractility induced by ozone. Dexamethasone inhibits ACh-induced ASM contraction through phosphorylation of p38 MAPK and HSP27