Prolonged cytopenias after immune effector cell therapy and lymphodepletion in patients with leukemia, lymphoma and solid tumors.

BACKGROUND AIMS: The success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other malignancies. Although the efficacy of CAR T-cells is enhanced when administered post-lymphodepleting chemotherapy, this can trigger bone marrow suppression and sustained cytopenia after CD19.CAR T-cell therapy. Additionally, systemic inflammation associated with CAR T-cell activity may contribute to myelosuppression. Cytopenias, such as neutropenia and thrombocytopenia, elevate the risk of severe infections and bleeding, respectively. However, data on the incidence of prolonged cytopenias after immune effector therapy in the solid tumor context remain limited. OBJECTIVE: We compared the incidence of prolonged cytopenias after immune effector therapy including genetically modified T-cells, virus-specific T-cells (VSTs) and NKT-cells, as well non-gene-modified VSTs for leukemia, lymphoma, and solid tumors (ST) to identify associated risk factors. METHODS: A retrospective analysis was conducted of 112 pediatric and adult patients with relapsed and/or refractory cancers who received lymphodepleting chemotherapy followed by immune effector therapy. Patients treated with 13 distinct immune effector cell therapies through 11 single-center clinical trials and 2 commercial products over a 6-year period were categorized into 3 types of malignancies: leukemia, lymphoma and ST. We obtained baseline patient characteristics and adverse events data for each participant, and tracked neutrophil and platelet counts following lymphodepletion. RESULTS: Of 112 patients, 104 (92.9%) experienced cytopenias and 88 (79%) experienced severe cytopenias. Patients with leukemia experienced significantly longer durations of severe neutropenia (median duration of 14 days) compared with patients with lymphoma (7 days) or ST (11 days) (P = 0.002). Patients with leukemia also had a higher incidence of severe thrombocytopenia (74.1%), compared with lymphoma (46%, P = 0.03) and ST (14.3%, P < 0.0001). Prolonged cytopenias were significantly associated with disease type (63% of patients with leukemia, 44% of patients with lymphoma, and 22.9% of patients with ST, P = 0.006), prior hematopoietic stem cell transplant (HSCT) (66.7% with prior HSCT versus 38.3% without prior HSCT, P = 0.039), and development of immune effector cell-associated neurotoxicity syndrome (ICANS) (75% with ICANS versus 38% without ICANS, P = 0.027). There was no significant association between prolonged cytopenias and cytokine release syndrome. CONCLUSIONS: Immune effector recipients often experience significant cytopenias due to marrow suppression following lymphodepletion regardless of disease, but prolonged severe cytopenias are significantly less common after treatment of patients with lymphoma and solid tumors.

A fingerprint of 2-[18F]FDG radiometabolites - How tissue-specific metabolism beyond 2-[18F]FDG-6-P could affect tracer accumulation.

Studies indicate that the radiotracer 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) can be metabolized beyond 2-[18F]FDG-6-phosphate (2-[18F]FDG-6-P), but its metabolism is incompletely understood. Most importantly, it remains unclear whether downstream metabolism affects tracer accumulation in vivo. Here we present a fingerprint of 2-[18F]FDG radiometabolites over time in cancer cells, corresponding tumor xenografts and murine organs. Strikingly, radiometabolites representing glycogen metabolism or the oxPPP correlated inversely with tracer accumulation across all examined tissues. Recent studies suggest that not only hexokinase, but also hexose-6-phosphate dehydrogenase (H6PD), an enzyme of the oxidative pentose phosphate pathway (oxPPP), determines 2-[18F]FDG accumulation. However, little is known about the corresponding enzyme glucose-6-phosphate dehydrogenase (G6PD). Our mechanistic in vitro experiments on the role of the oxPPP propose that 2-[18F]FDG can be metabolized via both G6PD and H6PD, but data from separate enzyme knockdown suggest diverging roles in downstream tracer metabolism. Overall, we propose that tissue-specific metabolism beyond 2-[18F]FDG-6-P could matter for imaging.

Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins.

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

If It Works, Don't Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism.

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells' metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells' metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation.

Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.

Monocytes exposed to plasma from patients with Alzheimer's disease undergo metabolic reprogramming.

The search for a blood-based biomarker that identifies Alzheimer's disease (AD) and can replace current invasive and expensive diagnostic tests, continues. The most extensively-examined peripheral marker is ß-amyloid (Aß) but the results are inconsistent across studies and do not reflect the changes that take place in the brain. Several studies have assessed possible proteomic signatures but with inconsistent findings, although increases in circulating inflammatory molecules are generally observed. Here, rather than focus on identifying changes in the circulation, we evaluated the effect of plasma from patients with mild cognitive impairment (MCI) and AD on the human monocyte-like cell line, THP-1 cells, and plasma from an AD mouse model on a mouse monocyte-macrophage cell line, J774.2 cells. Plasma from AD patients and the AD mouse model increased inflammatory molecules in the cells and these changes were accompanied by an increase in glycolysis. Interestingly, plasma from MCI patients exerted no significant effect on THP-1 cells. The possibility therefore exists that evaluating the effect of plasma on IL-8 and TNFα mRNA in THP-1 cells combined with analysis of glycolysis in these cells, may be the basis of an indicator that discriminates between AD and MCI and normal controls, but is unlikely to be useful in identifying early pathological changes.

Implications of the diagnostic criteria of idiopathic pulmonary fibrosis in clinical practice: Analysis from the Australian Idiopathic Pulmonary Fibrosis Registry.

BACKGROUND AND OBJECTIVE: Current guidelines for the diagnosis of idiopathic pulmonary fibrosis (IPF) provide specific criteria for diagnosis in the setting of multidisciplinary discussion (MDD). We evaluate the utility and reproducibility of these diagnostic guidelines, using clinical data from the Australian IPF Registry. METHODS: All patients enrolled in the registry undergo a diagnostic review whereby international IPF guidelines are applied via a registry MDD. We investigated the clinical applicability of these guidelines with regard to: (i) adherence to guidelines, (ii) Natural history of IPF diagnostic categories and (iii) Concordance for diagnostic features. RESULTS: A total of 417 participants (69% male, 70.6 ± 8.0 years) with a clinical diagnosis of IPF underwent MDD. The 23% of participants who did not meet IPF diagnostic criteria displayed identical disease behaviour to those with confirmed IPF. Honeycombing on radiology was associated with a worse prognosis and this translated into poorer prognosis in the 'definite' IPF group. While there was moderate agreement for IPF diagnostic categories, agreement for specific radiological features, other than honeycombing, was poor. CONCLUSION: In clinical practice, physicians do not always follow IPF diagnostic guidelines. We demonstrate a cohort of IPF patients who do not meet IPF diagnostic guideline criteria, based largely on their radiology and lack of lung biopsy, but who have outcomes identical to those with IPF.

Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris.

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e-) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e-) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.

The quantitative link of lung clearance index to bronchial segments affected by bronchiectasis.

In adult patients with cystic fibrosis (CF), the lung clearance index (LCI) derived from the multiple breath washout relates to both acinar and conductive ventilation heterogeneity. The latter component predicts an association between LCI and the number of bronchial segments affected by bronchiectasis. Here, we experimentally demonstrated this association in patients with CF, and also examined an ancillary group of patients with non-CF bronchiectasis. We conclude that lung disease severity in terms of number of bronchial segments results in an associated LCI increase, likely constituting a portion of LCI that cannot be reversed by treatment in patients with CF lung disease.

Exploring Metabolic Configurations of Single Cells within Complex Tissue Microenvironments.

Over the past years, plenty of evidence has emerged illustrating how metabolism supports many aspects of cellular function and how metabolic reprogramming can drive cell differentiation and fate. Here, we present a method to assess the metabolic configuration of single cells within their native tissue microenvironment via the visualization and quantification of multiple enzymatic activities measured at saturating substrate conditions combined with subsequent cell type identification. After careful validation of the approach and to demonstrate its potential, we assessed the intracellular metabolic configuration of different human immune cell populations in healthy and tumor colon tissue. Additionally, we analyzed the intercellular metabolic relationship between cancer cells and cancer-associated fibroblasts in a breast cancer tissue array. This study demonstrates that the determination of metabolic configurations in single cells could be a powerful complementary tool for every researcher interested to study metabolic networks in situ.

Short-term cooling increases serum triglycerides and small high-density lipoprotein levels in humans.

BACKGROUND: Cold exposure and ß3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase-mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. OBJECTIVE: The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. METHODS: Body mass index-matched young, lean men were exposed to a personalized cooling protocol for 2 hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1H-nuclear magnetic resonance. Adenosine triphosphate-binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [3H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. RESULTS: Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low-density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro. CONCLUSIONS: Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

Implementing goal-directed protocols reduces length of stay after cardiac surgery.

OBJECTIVE: To test the effect of a high reliability organization (HRO) intervention on patient lengths of stay in the CVICU and hospital. The authors proposed that (1) higher safety related evidence based protocol (SREBP) team compliance scores and (2) lower SREBP milestone scores are associated with shorter lengths of CVICU and hospital stay. DESIGN: A prospective, longitudinal observational evaluation was used to assess the effects of SREBP-focused rounding processes and a milestone-tracking tool. SETTING: United States, university academic medical center's 27-bed CVICU. PARTICIPANTS: Six hundred sixty-five adult cardiac surgery patients and the CVICU care team (100 registered nurses and 16 clinical providers) participated. MEASUREMENTS AND MAIN RESULTS: Team compliance was the proportion of SREBP-related team behaviors exhibited during daily rounds. Patients' milestone scores were the cumulative difference between actual and expected times for 4 SREBP milestones over 48 hours. Milestones achieved earlier than expected indicated reduced complication risk, and milestones achieved later than expected indicated increased risk. As team compliance increased, CVICU length of stay decreased 0.66 (95% CI: -0.04 to 1.28; p = 0.08) days; hospital stay decreased 0.89 times (95% CI: 0.77-1.03; p = 0.008). As the mean milestone scores increased from -7 to 12, length of ICU stay increased 2.63 (95% CI: 1.66-3.59; p<0.001) days; hospital length of stay increased 1.44 times (95% CI: 1.23-1.7; p = 0.05). CONCLUSIONS: A milestone-driven pathway supported by team rounding was associated with decreased lengths of CVICU and hospital stay. However, tracking patient trajectories by milestones suggests a more complex relationship than anticipated and presents new opportunities for SREBP implementation and research.

Active axial spondyloarthritis: potential role of certolizumab pegol.

The axial spondyloarthropathies are a group of chronic inflammatory diseases that predominantly affect the axial joints. This group includes ankylosing spondylitis and nonradiographic axial spondyloarthropathy. While the pathogenesis of axial spondyloarthropathies is not clear, immunologically active tissues primarily include the entheses, ie, the areas where ligaments, tendons, and joint capsules attach to bone and to the annulus fibrosis at the vertebrae. One of the major mediators of the immune response in this group of diseases is tumor necrosis factor-alpha (TNFα). Blockade of TNFα results in reduced vascularity and inflammatory cell infiltration in the synovial tissues of affected joints. Certolizumab pegol (CZP) is an Fc-free, PEGylated anti-TNFα monoclonal antibody. CZP has unique properties that differ from other available TNFα inhibitors by virtue of its lack of an Fc region, which minimizes potential Fc-mediated effects, and its PEGylation, which improves drug pharmacokinetics and bioavailability. It has been shown in clinical trials that CZP improves patient outcomes and reduces inflammation in the sacroiliac joints and spine in both ankylosing spondylitis and nonradiographic axial spondyloarthropathies. These data support CZP as a treatment option for axial spondyloarthropathies.

Ether bridge formation in loline alkaloid biosynthesis.

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine.

Is rheumatoid factor useful in primary care? A retrospective cross-sectional study.

Rheumatoid factor (RF) is frequently tested in general practice where its utility as a diagnostic test for rheumatoid arthritis (RA) is not known. We undertook a retrospective cross-sectioal study to determine the utility and cost of RF in a primary care population. We compared RF with recorded clinical features based on the American College of Rheumatology (ACR) criteria as a diagnostic test for RA in 235 patients in general practice using receiver operating characteristic curves and calculated the cost of testing per case of RA. We analysed 36,191 RF requests made to one laboratory from 2003-2009 at a mean annual cost of £58,164 and the variation and annual cost of RF testing between 77 practices. The sensitivity and specificity of RF at the optimal cut-off value of 20 U/ml were 0.6 and 0.96 and that of two documented clinical ACR criteria were 0.9 and 0.92, respectively. No ACR criteria were documented in 150 (63.8%) patients who had RF tested. The overall cost of RF testing per case of seropositive RA was £708.75. Of all RF requests, 66.6% was made by GPs, 7.0% by rheumatologists and 26.4% by other hospital departments. The proportion of positive tests was 5.8% in primary care and 17.7% in rheumatology. The mean number of tests performed annually in primary care was 4.65 (SD 2.7) per 1,000 patients. RF is less sensitive for RA than clinical features in primary care and is frequently requested in cases without clinical evidence of the disease, adding to the overall cost.

Geometric and electronic structures of manganese-substituted iron superoxide dismutase.

The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N-Mn-N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (E(m)) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry 2001, 40, 13079-13087), E(m)'s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated E(m) of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Immunotherapies in rheumatologic disorders.

Over the past several decades, rheumatology has directed its focus to understanding and countering the immune dysregulation underlying autoimmune diseases with rheumatologic manifestations. Older therapies, effective though poorly understood, are being scrutinized anew and are yielding the immune-modulating mechanisms behind their efficacy. New therapies, the "biologics," are drugs tailored to address specific immune defects and imbalances. This article discusses the current standard and biologic immunotherapies of the rheumatic diseases, correlating our current understanding of their mechanisms with dysfunctions believed to be present in the major autoimmune syndromes, especially rheumatoid arthritis and systemic lupus erythematosus.