Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression, and memory-related behaviors.

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. Here, we show, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene deletion) and pharmacological (treatment with the MIF antagonist Iso-1) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

La cresta alveolar atrófica en implantología oral

Los implantes dentales endoóseos constituyen una alternativa terapéutica ideal en pacienfes parcial o totalmente edéntulos; sin embargo, en ocasiones, la atrofia ósea generalizada o localizada contraindica el tratamiento, siendo necesario realizar un aumento de cresta alveolar que permita, bien simultáneamente o deforma diferida, la adecuada rehabilitación implantológica. En el presente trabajo, hacemos una revisión de las distintas técnicas y materiales utilizados actualmente en implantología, para mejorar las condiciones de recepción de los implantes, en presencia de defectos óseos.

Hémorragie intra-alvéolaire [Alveolar hemorrhage].

Diffuse alveolar hemorrhage (DAH) is defined by the presence of red blood cells originating from the lung capillaries or venules within the alveoli. The diagnosis is established on clinical features, radiological pattern, and especially bronchoalveolar lavage. Diffuse alveolar hemorrhage may have many immune or non-immune causes. Immune causes of DAH include vasculitides, connective tissue diseases, especially systemic lupus erythematosus, and antiglomerular basement membrane antibody disease (Goodpasture's syndrome). Treatment is both supportive and causal, often based on high dose corticosteroids and immunosuppressive therapy (especially intravenous cyclophosphamide). Plasma exchanges are performed in antiglomerular basement membrane antibody disease and systemic lupus erythematosus, and are considered in systemic vasculitis. Non-immune causes of DAH mainly include heart diseases, coagulation disorders, infections, drug toxicities and idiopathic DAH. Treatment of non-immune DAH is that of its cause. Whatever the cause, DAH is an emergency requiring prompt assessment and early treatment.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Surfactant palmitoylmyristoylphosphatidylcholine is a marker for alveolar size during disease.

Two common lung-related complications in the neonate are respiratory distress syndrome, which is associated with a failure to generate low surface tension at the air-liquid interface because of pulmonary surfactant insufficiency, and bronchopulmonary dysplasia (BPD), a chronic lung injury with reduced alveolarization. Surfactant phosphatidylcholine (PC) molecular species composition during alveolarization has not been examined. Mass spectrometry analysis of bronchoalveolar lavage fluid of rodents and humans revealed significant changes in surfactant PC during alveolar development and BPD. In rats, total PC content rose during alveolarization, which was caused by an increase in palmitoylmyristoyl-PC (16:0/14:0PC) concentration. Furthermore, two animal models of BPD exhibited a specific reduction in 16:0/14:0PC content. In humans, 16:0/14:0PC content was specifically decreased in patients with BPD and emphysema compared with patients without alveolar pathology. Palmitoylmyristoyl-PC content increased with increasing intrinsic surfactant curvature, suggesting that it affects surfactant function in the septating lung. The changes in acyl composition of PC were attributed to type II cells producing an altered surfactant during alveolar development. These data are compatible with extracellular surfactant 16:0/14:0PC content being an indicator of alveolar architecture of the lung.

Susceptibility of clinical isolates of Leishmania aethiopica to miltefosine, paromomycin, amphotericin B and sodium stibogluconate using amastigote-macrophage in vitro model.

Cutaneous Leishmaniasis (CL) caused by Leishmania aethiopica is a public health and social problem with a sequel of severe and mutilating skin lesions. It is manifested in three forms: localized CL (LCL), mucosal CL (MCL) and diffuse CL (DCL). Unresponsiveness to sodium stibogluconate (Sb(V)) is common in Ethiopian CL patients. Using the amastigote-macrophage in vitro model the susceptibility of 24 clinical isolates of L. aethiopica derived from untreated patients was investigated. Eight strains of LCL, 9 of MCL, and 7 of DCL patients together with a reference strain (MHOM/ET/82/117/82) were tested against four antileishmanial drugs: amphotericin B, miltefosine, Sb(V) and paromomycin. In the same order of drugs, IC(50) (μg/ml±SD) values for the 24 strains tested were 0.16±0.18, 5.88±4.79, 10.23±8.12, and 13.63±18.74. The susceptibility threshold of isolates originating from the 3 categories of patients to all 4 drugs was not different (p>0.05). Maximal efficacy was superior for miltefosine across all the strains. Further susceptibility test could validate miltefosine as a potential alternative drug in cases of sodium stibogluconate treatment failure in CL patients.

The role of macrophage migration inhibitory factor in mouse islet transplantation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. METHODS: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. RESULTS: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. CONCLUSION: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

Increased macrophage migration inhibitory factor (MIF) plasma levels in acute HIV-1 infection.

Considering macrophage migratory inhibitory factor (MIF) as a critical pro-inflammatory cytokine of the immune system, we evaluated plasma MIF levels in 89 HIV-infected adults. Plasma MIF levels were higher in HIV-infected than in HIV-negative individuals. Highest MIF levels were observed during acute HIV infection (AHI) whilst patients on antiretroviral therapy (ART) had lower MIF levels, regardless of ART efficacy. Our results suggest that MIF is an integral component of the cytokine storm characteristic of AHI.