Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

<p>Abstract</p> <p>Background</p> <p>Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats.</p> <p>Results</p> <p>POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin.</p> <p>Conclusion</p> <p>Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.</p

Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5−/− Mice

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5(-/-) mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5(-/-) animals tested previously in similar experimental setups

Knock-in mice expressing a 15-lipoxygenating Alox5 mutant respond differently to experimental inflammation than reported mice

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5(-/-) mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5(-/-) animals tested previously in similar experimental setups

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Background Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. Results In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. Conclusion Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated

A cross-sectional study to estimate the point prevalence of painful diabetic neuropathy in Eastern Libya.

BACKGROUND: Painful Diabetic Neuropathy (PDN) is a complication that affects up to one third of people living with diabetes. There is limited data on the prevalence of PDN from countries in the Middle East and North Africa. The aim of this study was to estimate the point prevalence of PDN in adults in Eastern Libya using the self-report Leeds Assessment of Neuropathic Symptoms and Signs (S-LANSS) pain scale. METHODS: We invited patients attending the Benghazi Diabetes Centre who had diabetes for ≥ 5 years to take part in the study. Patients provided consent and completed the Arabic S-LANSS. Anthropometrics, marital status, socioeconomic and education information was recoded and fasting plasma glucose concentration determined. RESULTS: Four hundred and fifty participants completed the study (age = 19 to 87 years, BMI = 17.6 to 44.2 kg/m2, 224 women). One hundred and ninety five participants (43.3%) reported pain in their lower limbs in the previous 6 months and 190/195 participants (97.4%) reported a S-LANSS score of ≥ 12 suggesting they had neuropathic pain characteristics. Thus, 42.2% (190/450) of participants with diabetes were categorised as experiencing pain with neuropathic characteristics. Mean ± SD duration of diabetes for participants with PDN (20.4 ± 6.5 years) was significantly higher compared with those without PDN (11.1 ± 4.6 years). Participants with PDN smoked tobacco for more years than those without pain (7.9 ± 12.3 years versus 1.1 ± 3.9 years respectively); had significantly higher fasting plasma glucose concentration (143.6 ± 29.3 mg/dl versus 120.0 ± 17.3 mg/dl) and had a significantly higher levels of education and employment status. The most significant predictors of PDN were duration of diabetes (OR = 25.85, 95% CI = 13.56-49.31), followed by smoking for men (OR = 8.28, 95% CI = 3.53-9.42), obesity (OR = 3.96, 95% CI = 2.25-6.96) and high fasting plasma glucose concentration (OR = 3.51, 95% CI = 1.99-6.21). CONCLUSION: The prevalence of PDN in people with diabetes in Eastern Libya was 42.2%. Risk factors for developing PDN were high fasting plasma glucose concentration, long duration of diabetes, and higher level of educational and employment status

mu-Opioid receptor antibody reveals tissue-dependent specific staining and increased neuronal mu-receptor immunoreactivity at the injured nerve trunk in mice

Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal mu-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the mu-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the mu-receptor antibody staining specificity. We found that the antibody specifically labeled mu-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of mu/delta/kappa-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of mu-receptor expression in the DRG was not possible. However, we demonstrate that the mu-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, mu-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies' specificity, preferably using knockout animals