Tissue-protective cytokines

While many cytokines are known for their inflammatory action, there is a growing interest in the tissue-protective effects of some cytokines. The prototypic tissue-protective cytokine is EPO. Initially described as neuro-protective, it is beneficial in animal models of ischemic and other types of injury. Scientists had to overcome the notion that EPO had only erythropoietic actions, was only produced by the kidney, and that its receptor was only present in erythroid progenitor cells. The use of in vitro and in vivo disease models was essential to demonstrate the protective effects of EPO. Reproducible models will be needed for the further study of the mechanism of action of EPO and for the identification of other tissue-protective cytokines. In Tissue-Protective Cytokines: Methods and Protocols, expert researchers in the field detail the key models that have been used to characterize the tissue-protective actions of cytokines. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, provide step-by-step laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Thorough and intuitive, Tissue Protective Cytokines: Methods and Protocols aids scientists in continuing to study tissue-protection that will be a new field of interest of cytokine biology, both in discovering novel actions of known cytokines and in developing new drugs

Relief of allodynia depends upon the ß-common receptor (ßcR).

<p><b>A.</b> Both ketamine and ARA 290 did not prevent the development of allodynia in ßcR^−/− mice. <b>B.</b> However, the effect of ketamine on nociceptive pain is unchanged in ßcR^−/− animals (treatment effect, p<0.001).</p

Ketamine and ARA 290 have similar effects on allodynia.

<p><b>A.</b> Treatment with both ketamine and ARA 290 prevented the full development of allodynia (treatment effect, p = 0.049 and p = 0.03, respectively). <b>B.</b> The effects of ketamine on acute nociceptive pain remained unchanged over time (treatment effect, p<0.001).</p

Neuropathic pain involves a pathway that utilizes the Innate Immune Receptor.

<p>Nerve injury results in microglial recruitment, increased expression of NMDAR, and proinflammatory cytokine production, ultimately resulting in allodynia. Activation of the innate immune receptor (IRR), e.g., by ARA 290, antagonizes this pathway. Ketamine also requires the IRR to reduce allodynia. This may be via a direct interaction with the IRR or alternatively, via modulation of intermediate processes that are upstream of the IRR. Additionally, ketamine interacts with NMDARs that mediate antinociception and psychomotor effects. ARA 290 does not interact with the NMDAR and therefore lacks these additional effects.</p

Ketamine and ARA 290 differ in effects on acute nociceptive pain and side effects.

<p><b>A.</b> Ketamine administration increases the latency of tail withdrawal to a thermal stimulus (treatment effect, p<0.001), whereas ARA 290 does not. <b>B.</b> Ketamine treatment had significant biphasic effects on stereotypic behavior: after a period of transient sedation, the animals showed signs of psychomimetic disturbances that lasted for about 20 minutes (treatment effect, p<0.001). ARA 290 did not display these side effects. <b>C.</b> Treatment with ketamine was associated with a biphasic activation of generalized activity (treatment effect, p<0.001) causing an increase in restlessness and explorative behavior after a period of transient sedation.</p

Ketamine and ARA 290 reduce inflammatory mediators in the spinal cord following sciatic nerve injury.

<p>One week post surgery, animals showed a marked elevation of CCL2 (panel <b>A</b>), Iba1 (panel <b>B</b>), and GFAP (panel <b>C</b>) compared to naïve controls. Both ketamine and ARA 290 significantly reduced the mRNA levels of these genes to a similar extent. *p<0.05 <i>versus</i> vehicle, #p<0.05 between ketamine and ARA 290 treatments, **p<0.05 <i>versus</i> naïve.</p

QRT-PCR primers and probes used in this study.

<p>Primers and probes used for the quantification of mRNA from NMDA receptor subtypes NR1, NR2A and NR2B (Grin); microglia marker Iba-1 (AIF-1), astrocyte (GFAP) and CCL2; f, Reporter dye1 (FAM:6-carboxyfluorescein); t, Reporter dye2 (TET:Tetrachloro-6-carboxyfluorescein); q, Quencher dye (TAMRA: 6-carboxytetramethyl1-rhodamine).</p

Ketamine and ARA 290 reduce mRNA for NDMA receptor subunits in established neuropathy.

<p>Real time PCR data show that NMDA receptor subunits 1 (panel <b>A</b>), 2A (panel <b>B</b>), and 2B (panel <b>C</b>) are all modestly elevated one week following sciatic nerve injury. Administration of ketamine significantly reduces mRNA to baseline levels. In contrast, ARA 290 reduced mRNA for these receptor subunits to substantially below baseline (naïve). *p<0.05 <i>versus</i> vehicle, #p<0.05 between ketamine and ARA 290 treatments, **p<0.05 <i>versus</i> naïve.</p

Data_Sheet_1_A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.docx

BackgroundAging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age?MethodsWe conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan–Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions.ResultsChronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life.ConclusionAdministration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.</p