Disentangling acceleration-, velocity-, and duration-dependency of the short- and medium-latency stretch reflexes in the ankle plantarflexors

Previous research and definitions of the stretch reflex and spasticity have focused on velocity dependence. We showed that perturbation acceleration, velocity, and duration all shape the M1 and M2 response, often via nonlinear or interacting dependencies. Consequently, systematic execution and reporting of stretch reflex and spasticity studies, avoiding uncontrolled parameter interdependence, is essential for proper understanding of the reflex neurophysiology.

Anti-Inflammatory (M2) Response Is Induced by a sp2-Iminosugar Glycolipid Sulfoxide in Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most common complications of Diabetes Mellitus (DM) and is directly associated with inflammatory processes. Currently, neuro-inflammation is considered an early event in DR and proceeds via microglia polarization. A hallmark of DR is the presence of retinal reactive gliosis. Here we report the beneficial effect of (SS,1R)-1-docecylsulfiny-5N,6O-oxomethylidenenojirimycin ((Ss)-DS-ONJ), a member of the sp2-iminosugar glycolipid (sp2-IGL) family, by decreasing iNOS and inflammasome activation in Bv.2 microglial cells exposed to pro-inflammatory stimuli. Moreover, pretreatment with (Ss)-DS-ONJ increased Heme-oxygenase (HO)-1 as well as interleukin 10 (IL10) expression in LPS-stimulated microglial cells, thereby promoting M2 (anti-inflammatory) response by the induction of Arginase-1. The results strongly suggest that this is the likely molecular mechanism involved in the anti-inflammatory effects of (SS)-DS-ONJ in microglia. (SS)-DS-ONJ further reduced gliosis in retinal explants from type 1 diabetic BB rats, which is consistent with the enhanced M2 response. In conclusion, targeting microglia polarization dynamics in M2 status by compounds with anti-inflammatory activities offers promising therapeutic interventions at early stages of DR.

TheROP16III-dependent early immune response determines the sub-acute CNS immune response and type IIIToxoplasma gondiisurvival

Toxoplasma gondiiis an intracellular parasite that persistently infects the CNS and that has genetically distinct strains which provoke different acute immune responses. How differences in the acute immune response affect the CNS immune response is unknown. To address this question, we used two persistentToxoplasmastrains (type II and type III) and examined the CNS immune response at 21 days post infection (dpi). Contrary to acute infection studies, type III-infected mice had higher numbers of total CNS T cells and macrophages/microglia but fewer alternatively activated macrophages (M2s) and regulatory T cells (Tregs) than type II-infected mice. By profiling splenocytes at 5, 10 and 21 dpi, we determined that at 5 dpi type III-infected mice had more M2s while type II-infected mice had more classically activated macrophages (M1s) and these responses flipped over time. To test how these early differences influence the CNS immune response, we engineered the type III strain to lack ROP16 (IIIΔrop16), the polymorphic effector protein that drives the type III-associated M2 response. IIIΔrop16-infected mice showed a type II-like neuroinflammatory response with fewer infiltrating T cells and macrophages/microglia and more M2s and an unexpectedly low CNS parasite burden. At 5 dpi, IIIΔrop16-infected mice showed a mixed inflammatory response with more M1s, M2s, T effector cells, and Tregs, and decreased rates of infection of peritoneal exudative cells (PECs). These data suggested that type III parasites need the early ROP16-associated M2 response to avoid clearance, possibly by the Immunity-Related GTPases (IRGs), IFN-γ dependent proteins essential for murine defenses againstToxoplasma. To test this possibility, we infected IRG-deficient mice and found that IIIΔrop16parasites now maintained parental levels of PECs infection. Collectively, these studies suggest that, for the type III strain,rop16IIIplays a key role in parasite persistence and influences the sub-acute CNS immune response.Author SummaryToxoplasmais a ubiquitous intracellular parasite that establishes an asymptomatic brain infection in immunocompetent individuals. However, in the immunocompromised and the developing fetus,Toxoplasmacan cause problems ranging from fever to chorioretinitis to severe toxoplasmic encephalitis. Emerging evidence suggests that the genotype of the infectingToxoplasmastrain may influence these outcomes, possibly through the secretion ofToxoplasmastrain-specific polymorphic effector proteins that trigger different host cell signaling pathways. While such strain-specific modulation of host cell signaling has been shown to affect acute immune responses, it is unclear how these differences influence the sub-acute or chronic responses in the CNS, the major organ affected in symptomatic disease. This study shows that genetically distinct strains ofToxoplasmaprovoke strain-specific CNS immune responses and that, for one strain (type III), the acute and sub-acute immune responses and parasite survival are heavily influenced by a polymorphic parasite gene (rop16III).

Abstract T P80: Inhibition of Ezh2 Leads to Decreased M1 and Enhanced M2 Microglia Phenotypes

Background: Ischemic stroke results in the activation of microglia, which may polarize toward a pro-inflammatory (M1) phenotype or an anti-inflammatory, neuroprotective (M2) phenotype. Thus, simultaneously suppressing the M1 response and promoting the M2 response could be beneficial in the treatment of stroke. Recently, the epigenetic modulator Jmjd3 has been shown to be essential for M2 polarization. However, Jmjd3 is antagonized by Ezh2 which is associated with M1 polarization. Thus, we hypothesized that inhibition of Ezh2 tilts the balance between Jmjd3 and Ezh2, thereby enhancing polarization toward an M2 phenotype and improved outcome in ischemic stroke. Methods: Mixed glial cultures were isolated from P0.5-P2 C57BL/6J mice and cultured for 14 days before microglial isolation. Microglia were rested for 24 hours before treatment every other day with 6uM GSK343 (Cayman Chemical) or DMSO vehicle control. After 7 days, microglia were stimulated with LPS or IL-4 and RNA was isolated at 4hr and 24hr post-stimulation for qRT-PCR analysis. Results: LPS-induced IL6 and IL1B expression was significantly abrogated by 71% and 53%, respectively (p<0.05), at 24hr when Ezh2 was inhibited. Additionally, Ezh2 inhibition both increased baseline expression of M2-associated genes ARG1, CD206, and IRF4 by 196%, 257%, and 395%, respectively (p<0.05), and rescued their expression in the presence of LPS at 24hr (p<0.05) in which they were otherwise significantly down-regulated. Conclusion: Pharmacological inhibition of Ezh2 limits microglial M1 polarization and enhances M2 polarization.

Paraoxonase 2 Induces a Phenotypic Switch in Macrophage Polarization Favoring an M2 Anti-Inflammatory State

Inflammatory processes are involved in atherosclerosis development. Macrophages play a major role in the early atherogenesis, and they are present in the atherosclerotic lesion in two phenotypes: proinflammatory (M1) or anti-inflammatory (M2). Paraoxonase 2 (PON2) is expressed in macrophages, and it was shown to protect against atherosclerosis. Thus, the aim of our study was to analyze the direct effect of PON2 on macrophage inflammatory phenotypes. Ex vivo studies were performed with murine peritoneal macrophages (MPM) harvested from control C57BL/6 and PON2-deficient (PON2KO) mice. PON2KO MPM showed an enhanced proinflammatory phenotype compared to the control, both in the basal state and following M1 activation by IFNγand lipopolysaccharide (LPS). In parallel, PON2KO MPM also showed reduced anti-inflammatory responses in the basal state and also following M2 activation by IL-4. Moreover, the PON2-null MPM demonstrated enhanced phagocytosis and reactive oxygen species (ROS) production in the basal state and following M1 activation. The direct effect of PON2 was shown by transfecting human PON2 (hPON2) into PON2KO MPM. PON2 transfection attenuated the macrophages’ response to M1 activation and enhanced M2 response. These PON2 effects were associated with attenuation of macrophages’ abilities to phagocyte and to generate ROS. We conclude that PON2 promotes an M1 to M2 switch in macrophage phenotypes.

Experimental Pneumocystis lung infection promotes M2a alveolar macrophage-derived MMP12 production

Among several bacterial and viral pathogens, the atypical fungal organism Pneumocystis jirovecii has been implicated as a contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD). In a previous study, we reported that Pneumocystis-colonized HIV-positive subjects had worse obstruction of airways and higher sputum levels of macrophage elastase/matrix metalloproteinase 12 (MMP12), a protease strongly associated with the development of COPD. Here, we examined parameters of Pneumocystis-induced MMP12 in the lungs of mice and its role in the lung immune response to murine Pneumocystis. Initial studies demonstrated that P. murina exposure induced Mmp12 mRNA expression in whole lungs and alveolar macrophages (AMs), which was dependent on the presence of CD4+ T cells as well as signal transducer and activator of transcription 6. Mmp12 mRNA expression was upregulated in AMs by interleukin (IL)-4 treatment, but downregulated by interferon (IFN)-γ, indicating preferential expression in alternatively activated (M2a) macrophages. IL-4 treatment induced the 54-kDa proenzyme form of MMP12 and the 22-kDa fully processed and active form, whereas IFN-γ failed to induce either. Despite a reported antimicrobial role in macrophage phagolysosomes, mice deficient in MMP12 were not found to be more susceptible to lung infection with P. murina. Collectively, our data indicate that MMP12 induction is a component of the P. murina-induced M2 response and thus provides insight into the link between Pneumocystis colonization/infection and exacerbations in COPD.

Interleukin-2 (IL-2) chrono-infusion (CHI) in metastatic renal cell carcinoma (mRCC): A phase I/II study.

423 Background: The soprachiasmatic nucleus represents the pacemaker coordinating circadian rhythm. Its dysregulation in neoplastic patients (pts) can be an important inducer/promoter of cancer progression. It is a potential mediator of cancer immunosuppressive effect and quality of life (QoL) impairment. CHI allows concentration of time active treatment with possible impact on QoL and immune and hormone set-up. Aim of this study was to evaluate feasibility, dose limiting toxicity and activity of CHI IL-2 in mRCC. Methods: 22 pts were enrolled, 14 were naive and 8 pretreated (chemo/immunotherapy in 6 and targeted in 6). M/F ratio was 16/6, median age 68 y. IL-2 CHI was administered iv in 8 h (peak 1 am, 9 am, 5 pm) from 2 MUI/m2 with escalated dose ( Fibonacci scale) x 3 days/q 2 wks for 4 cycles. Four maintenance cycles were performed in responsive/stable pts. Results: from January 2005 to July 2009, out of 22 pts, 3 were treated onto each Le (I-V ) and 7 in the VI. G3-4 toxicity was reported in four pts (hypotension in 2, renal in 2 and diarrhoea in 1) on the VI Le ( DLT 18,6 MUI/m2). Response: 1 CR, 2 PR, 9 SD, 10 PD. ORR was 25%%. DCR on overall/ good-intermediate group (MSKCC) was 54%/69%. Median PFS and OS were 4,5 and 14,5 mos respectively. In a univariate analysis dose Le (4-6 vs. 1-3), PS (0 vs 1-2), gender (M vs F) and cD3, cD4, cD8 count (gain vs drop) were statistically significant for OS. In a univariate analysis age (< 65 y vs. >65y), Motzer score (0-1-2 vs 3) increase/reduction of cD19, cD16, HLA-DR, cD4/cD8, increase/reduction of prolactine, reduction/increase of ACTH-Cortisol showed a trend of survival improvement. Conclusions: IL-2 CHI aappeared safe in a standard care unit, moderately toxic and active in mRCC. Phase 2 study with dose Le V is now ongoing.