Anne Camille La Flamme

A pilot study investigating the effects of remote ischemic preconditioning in high-risk cardiac surgery using a randomised controlled double-blind protocol

The efficacy of remote ischemic preconditioning (RIPC) in high-risk cardiac surgery is uncertain. In this study, 96 adults undergoing high-risk cardiac surgery were randomised to RIPC (3 cycles of 5xa0min of upper-limb ischemia induced by inflating a blood pressure cuff to 200xa0mmHg with 5xa0min of reperfusion) or control. Main endpoints were plasma high-sensitivity troponin T (hsTNT) levels at 6 and 12xa0h, worst post-operative acute kidney injury (AKI) based on RIFLE criteria, and noradrenaline duration. hsTNT levels were log-normally distributed and higher with RIPC than control at 6-h post cross-clamp removal [810xa0ng/ml (IQR 527–1,724) vs. 634xa0ng/ml (429–1,012); ratio of means 1.41 (99.17% CI 0.92–2.17); P=0.04] and 12xa0h [742xa0ng/ml (IQR 427–1,700) vs. 514xa0ng/ml (IQR 356–833); ratio of means 1.56 (99.17% CI 0.97–2.53); P=0.01]. After adjustment for baseline confounders, the ratio of means of hsTNT at 6xa0h was 1.23 (99.17% CI 0.88–1.72; P=0.10) and at 12xa0h was 1.30 (99.17% CI 0.92–1.84; P=0.05). In the RIPC group, 35/48 (72.9%) had no AKI, 5/48 (10.4%) had AKI risk, and 8/48 (16.7%) had either renal injury or failure compared to the control group where 34/48 (70.8%) had no AKI, 7/48 (14.6%) had AKI risk, and 7/48 (14.6%) had renal injury or failure (Chi-squared 0.41; two degrees of freedom; Pxa0=xa00.82). RIPC increased post-operative duration of noradrenaline support [21xa0h (IQR 7–45) vs. 9xa0h (IQR 3–19); ratio of means 1.70 (99.17% CI 0.86–3.34); P=0.04]. RIPC does not reduce hsTNT, AKI, or ICU-support requirements in high-risk cardiac surgery.

Type II-activated macrophages suppress the development of experimental autoimmune encephalomyelitis

Treatment with immune complexes, which ligate Fcγ receptors (FcγRs), suppresses the development of experimental autoimmune encephalomyelitis (EAE). To determine the mechanism of action, we investigated how these immune complexes affected type II activation of macrophages (that is, exposure to immune complexes in a proinflammatory environment). Our results show that lower doses of interferon‐γ (IFN‐γ) were more effective at priming bone marrow‐derived macrophages (BMMφ) to produce more interleukin 10 (IL‐10) and less IL‐12p40 in response to lipopolysaccharide (LPS) and immune complexes compared with LPS alone. Moreover, at the lowest level of IFN‐γ (20u2009Uu2009ml−1), a significant downregulation in the surface expression of CD40, CD80 and PD‐L1 was observed in LPS and immune complex‐stimulated macrophages (that is, type II activated) than macrophages stimulated with LPS alone (that is, classically activated). Finally, treatment of mice with type II‐activated macrophages protected them from developing EAE, suggesting that administration of immune complexes is protective against EAE by inducing type II‐activated macrophages.

Naïve blood monocytes suppress T-cell function. A possible mechanism for protection from autoimmunity.

In certain disease context, cells of the monocyte/macrophage lineage are known to exhibit T‐cell suppressor function. However, whether naïve monocytes are also able to suppress T‐cell responses has not been previously investigated. In this study, we have discovered that CD11b+Ly6G− mononuclear cells in the blood of naïve mice are potent suppressors of T‐cell proliferation in vitro. The suppression of T‐cell proliferation requires cell‐cell contact and is partially dependent on nitric oxide production. Following the induction of experimental autoimmune encephalomyelitis in mice, the suppressor function of this blood CD11b+Ly6G− cell population is impaired. Therefore, blood CD11b+Ly6G− cells appear to be intrinsically suppressive and may have a key role in maintaining immune homoeostasis. Loss of this suppressive function may contribute to development of autoimmunity.

Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplate® analyser.

Factors affecting the Multiplate® assays analytical precision have not been well defined. We investigated the effect of methodological factors on the measurement of ADP-induced platelet aggregation using the Multiplate® assay. ADP-induced platelet aggregation was analysed in whole blood using the Multiplate® assay. We tested the reproducibility of measurement, the effect of different anticoagulants (hirudin, citrate and heparin) and the effect of time delay (15, 30, 45, 60, 120 and 180 minutes) between sampling and analysis in patients. The use of a manual calibrated pipette with the Multiplate® analyser was also tested. The mean coefficient of variation (CV) using the manufacturers recommended methods was 10.8u2009±u20098.7% (nu2009=u200930). When compared to hirudin (359.5u2009±u2009309 AU*min) the use of heparin (521.0u2009±u2009316 AU*min, pu2009=u20090.0015) increased platelet aggregation, while the use of sodium citrate (245.0u2009±u2009209 AU*min, pu2009=u20090.003) decreased the platelet aggregation (nu2009=u200920). The addition of CaCl2 to the citrate-anticoagulated blood resulted in platelet aggregation levels similar to hirudin. Platelet aggregation varied with time delay (nu2009=u200920). When compared to platelet aggregation at 30 minutes (391.1u2009±u2009283 AU*min), platelet aggregation was reduced at 60 minutes (335.2u2009±u2009251.6 AU*min, pu2009<u20090.05), 120 minutes (198.8u2009±u2009122.9 AU*min, pu2009<u20090.001) and 180 minutes (160.7u2009±u200992 AU*min, pu2009<u20090.001). The use of a manual calibrated pipette did not significantly reduce the mean CV in the assay (nu2009=u200920). Methodological factors such as the anticoagulant used and the time delay should be standardised where possible to reduce variability, and allow thresholds derived from one study to be comparable across multiple studies.

Peloruside A, an Antimitotic Agent, Specifically Decreases Tumor Necrosis Factor-α Production by Lipopolysaccharide-Stimulated Murine Macrophages

Peloruside A (peloruside) is a naturally occurring compound isolated from a New Zealand marine sponge that, like the anticancer drug paclitaxel, stabilizes microtubules and inhibits mitosis. Paclitaxel is known to induce a proinflammatory response in murine macrophages; whereas, peloruside has never been tested for its immunomodulatory effects in these cells. Although the antimitotic effects of the two drugs appear to be similar, we found that peloruside, unlike paclitaxel, does not induce murine macrophages to produce the proinflammatory mediators interleukin-12p40 (IL-12p40), tumor necrosis factor-α (TNF-α), and nitric oxide. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay confirmed that the absence of cytokine production was not caused by cytotoxicity in these nondividing cells. Additionally, there was no effect on unstimulated splenocytes; whereas, both compounds inhibited proliferation after concanalavin A (Con A) stimulation. Finally, there was a significant decrease in TNF-α and nitric oxide but not IL-12p40 when macrophages were cultured with lipopolysaccharide (LPS) and either paclitaxel or peloruside. These results suggest that peloruside may prove to be an effective anti-inflammatory treatment, since it does not induce the production of proinflammatory mediators yet can downregulate TNF-α and nitric oxide production by LPS-stimulated macrophages, as well as inhibit lymphocyte proliferation.

Synergistic interactions between peloruside A and other microtubule-stabilizing and destabilizing agents in cultured human ovarian carcinoma cells and murine T cells

PurposeMicrotubule-stabilizing agents are an important class of anticancer compounds. Peloruside A and laulimalide bind to a different site on the microtubule to taxoid site drugs such as paclitaxel (Taxol®), docetaxel (Taxotere®), ixabepilone (Ixempra®), the epothilones, and discodermolide. The purpose of this study was to examine the synergistic interactions of these drugs when given in combination in relation to the differences in their binding sites on the microtubule.MethodsHuman ovarian carcinoma cells (1A9 cells) and murine T cells were treated with different combinations of microtubule-stabilizing or destabilizing agents. The compounds were given individually and in combination, and the antiproliferative activity was assessed to calculate a combination index (CI) from the equation: CIxa0=xa0D1/Dx1xa0+xa0D2/Dx2 in which D1 and D2 are the concentrations of drug 1 and drug 2 that when given together give the same response as drug 1 and 2 alone (Dx1 and Dx2). Thus, a CI value of less than 1.0 indicates a synergistic effect between the two drugs in which the response to the two drugs given together is greater than the additive response of the two drugs if given on their own.ResultsAs anticipated from previous inxa0vitro studies, peloruside A and laulimalide did not synergize with each other. They also failed to synergize with the microtubule-destabilizing agents vinblastine and 2-methoxyestradiol. Peloruside A and laulimalide did, however, synergize with the epothilones, as had been previously shown, but not with docetaxel or discodermolide.ConclusionsCombining two microtubule-targeting agents with different binding sites does not guarantee a synergistic interaction in cells, and additional factors are likely to be involved. This study highlights the importance of preclinical testing of actual combinations of drugs before proceeding into clinical trials.

Type II-Activated Murine Macrophages Produce IL-4

Background Type II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro. Results We found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα−/− macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results. Conclusions Taken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.

Delaying the onset of experimental autoimmune encephalomyelitis with the microtubule-stabilizing compounds, paclitaxel and Peloruside A

The hallmark of autoimmunity is the activation and proliferation of autoreactive lymphocytes. Therefore, one potential strategy to treat autoimmunity is to target the proliferating autoreactive lymphocytes with antimitotic drugs. Paclitaxel and peloruside are two microtubule‐stabilizing drugs that halt cell proliferation by stabilizing microtubules in the G2/M phase of the cell cycle. C57BL/6 mice treated for 5 consecutive days with paclitaxel or peloruside had a reduced incidence and significantly delayed development of EAE, a mouse model of MS. Although paclitaxel and peloruside were effective at inhibiting T cell proliferation in vitro, paclitaxel was shown to be ineffective at preventing the proliferation of autoreactive T cells in vivo during the 5‐day treatment period. However, after the 5‐day treatment, the ability of splenocytes or LN cells to proliferate in vitro was reduced significantly, suggesting that drug treatment targeted late but not early proliferative events in the animal. Moreover, in paclitaxel‐treated, MOG‐immunized mice, there was a complete inhibition of the recruitment of myeloid cells (especially macrophages) to the peripheral lymphoid organs. These results indicate that microtubule‐stabilizing drugs are effective at reducing disease but require a prolonged exposure to paclitaxel in vivo to alter proliferation in the myeloid and lymphoid cell compartments.

Targeting innate receptors with MIS416 reshapes Th responses and suppresses CNS disease in a mouse model of multiple sclerosis.

Modification of the innate immune cell environment has recently been recognized as a viable treatment strategy for reducing autoimmune disease pathology. MIS416 is a microparticulate immune response modifier that targets myeloid cells, activating cytosolic receptors NOD2 and TLR9, and has completed a phase 1b/2a trial for the treatment of secondary progressive multiple sclerosis. Using a mouse model of multiple sclerosis, we are investigating the pathways by which activation of TLR9 and NOD2 may modify the innate immune environment and the subsequent T cell-mediated autoimmune responses. We have found that MIS416 has profound effects on the Th subset balance by depressing antigen-specific Th1, Th17, and Th2 development. These effects coincided with an expansion of specific myeloid subpopulations and increased levels of MIS416-stimulated IFN-γ by splenocytes. Additionally, systemic IFN-γ serum levels were enhanced and correlated strongly with disease reduction, and the protective effect of MIS416 was abrogated in IFN-γ-deficient animals. Finally, treatment of secondary progressive MS patients with MIS416 similarly elevated the levels of IFN-γ and IFN-γ–associated proteins in the serum. Together, these studies demonstrate that administration of MIS416, which targets innate cells, reshapes autoimmune T cell responses and leads to a significant reduction in CNS inflammation and disease.

Treatment with the antipsychotic agent, risperidone, reduces disease severity in experimental autoimmune encephalomyelitis.

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)4, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.