The saturable, carrier-mediated system capable of the brain-to-blood transport of small peptides with an N-terminal tyrosine was characterized. The rate of disappearance of intraventricularly injected iodinated peptide in the presence or absence of the inhibitor being tested was determined from formulas based on the residual radioactivity in the brains of mice after decapitation. The injection of 100 nmol/mouse of unlabeled N-Tyr-MIF-1 (TMIF) increased the half-time disappearance of 125I-TMIF (ITMIF) in the central nervous system (CNS) from 14.1 to 88.7 min (P less than 0.00005). Technetium, a substance transported out of the brain by the same system that transports iodine, was used as a control; the half-time disappearance of technetium pertechnetate was unaffected by unlabeled TMIF. With two related but distinct techniques, the maximum transport rate out of the CNS (Vmax) for TMIF was 0.266 nmol X g of brain per min (method 1) and 0.297 nmol X g-1 X min-1 (method 2), while the amount of unlabeled material needed to achieve 50% of Vmax (Km) was 15.2 nmol/g (method 1) and 15.1 nmol/g (method 2). The lack of effect of the tyrosinated fragments of TMIF as inhibitors indicates that TMIF is being transported in intact form. The Vmax for methionine enkephalin determined with labeled and unlabeled methionine enkephalin was 0.630 nmol X g-1 X min-1 and the Km was 24.95 nmol/g. Studies with the metabolic modulators furosemide, acetozolamide, reserpine, ouabain, and theophylline suggest that the system is sodium dependent and probably independent of ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemoattraction of leukocytes into the brain after induction of middle cerebral artery occlusion (MCAO) increases the lesion size and worsens disease outcome. Our previous studies demonstrated that partial MHC class II constructs can reverse this process. However, the potential application of pMHC to human stroke is limited by the need to rapidly match recipient MHC class II with the β1 domain of the pMHC construct. We designed a novel recombinant protein comprised of the HLA-DRα1 domain linked to MOG-35-55 peptide but lacking the β1 domain found in pMHC and treated MCAO after 4 h reperfusion in humanized DR2 mice. Infarct volumes were quantified after 96 h reperfusion and immune cells from the periphery and CNS were evaluated for expression of CD74 and other cell surface, cytokine and pathway markers. This study demonstrates that four daily treatments with DRα1-MOG-35-55 reduced infarct size by 40 % in the cortex, striatum and hemisphere, inhibited the migration of activated CD11b+CD45high cells from the periphery to the brain and reversed splenic atrophy. Furthermore, DRα1-MOG-35-55 bound to CD74 on monocytes and blocked both binding and downstream signaling of macrophage migration inhibition factor (MIF) that may play a key role in infarct development. The novel DRα1-MOG-35-55 construct is highly therapeutic in experimental stroke and could be given to all patients at least 4 h after stroke onset without the need for tissue typing due to universal expression of DRα1 in humans.

The extraocular muscle fibers of vertebrates can be classified into two categories: singly innervated fibers (SIFs) and multiply innervated fibers (MIFs). In monkeys, the motoneurons of SIFs lie within the oculomotor, trochlear, and abducens nucleus, whereas the motoneurons of MIFs appear in separate subgroups in the periphery of the classical nuclei borders. In the present study, we investigated the histochemical properties of SIF and MIF motoneurons by using combined tract-tracing and immunofluorescence techniques. In monkeys, SIF and MIF motoneurons of extraocular muscles were identified by tracer injections into the belly or the distal myotendinous junction of the medial or lateral rectus muscle. Alternatively, the motoneurons were identified by choline acetyltransferase immunostaining. These techniques were combined with the detection of histochemical markers for perineuronal nets, nonphosphorylated neurofilaments, parvalbumin, or cytochrome oxidase. The experiments revealed that the MIF motoneurons in the periphery of the motonuclei do not contain nonphosphorylated neurofilaments or parvalbumin and lack perineuronal nets. In contrast, SIF motoneurons express all markers at high intensity. Cytochrome oxidase immunostaining was found in both motoneuron populations. An additional population of motoneurons with "MIF properties" was identified within the boundaries of the abducens nucleus, which could represent the motoneurons innervating MIFs in the orbital layer of lateral rectus muscle. Our data provide evidence that SIF and MIF motoneurons, which can be correlated with twitch motoneurons and presumed non-twitch motoneurons, differ in their histochemical properties. The absence of perineuronal nets, nonphosphorylated neurofilaments, and parvalbumin may help to identify the homologous MIF motoneurons in other species, including humans.

Ethanol-induced liver injury is a complex process dependent upon the interaction of multiple cell types in the liver, as well as activation of the innate immune response. Increased expression of CYP2E1 in response to high concentrations of ethanol leads to greater production of cytotoxic ethanol metabolites, which in turn contribute to production of reactive oxygen species, oxidative stress, and ultimately, cell death. Necroptotic hepatocyte cell death in response to ethanol is mediated via a CYP2E1-dependent expression of receptor-interacting protein kinase 3 (RIP3), a key component of the necroptosome. In response to alarmins released during ethanol-induced necroptosis, the innate immune response is activated. Macrophage migration inhibitory factor (MIF), a pro-inflammatory multikine involved in many disease processes, is an essential component to this response to injury. MIF expression is increased during ethanol exposure via a CYP2E1-dependent pathway, likely contributing to an exacerbated innate immune response and chronic inflammation after chronic ethanol. This review will discuss the complex interactions between CYP2E1-dependent expression of RIP3 and MIF in the pathophysiology of chronic ethanol-induced liver injury.

Ozone is an oxidizing environmental pollutant that contributes significantly to respiratory health. Exposure to increased levels of ozone has been associated with worsening of symptoms of patients with asthma and COPD (chronic obstructive pulmonary disease). In the present study, we investigated the acute and chronic effects of ozone exposure-induced oxidative stress-related inflammation mechanics in mouse lung. In particular, we investigated the oxidative stress-induced effects on HDAC2 (histone deacetylase 2) modification and activation of the Nrf2 (nuclear factor erythroid-related factor 2) and HIF-1α (hypoxia-inducible factor-1α) signalling pathways. Male C57BL/6 mice were exposed to ozone (3 p.p.m.) for 3 h a day, twice a week for a period of 1, 3 or 6 weeks. Control mice were exposed to normal air. After the last exposure, mice were killed for BAL (bronchoalveolar lavage) fluid and lung tissue collection. BAL total cell counts were elevated at all of the time points studied. This was associated with increased levels of chemokines and cytokines in all ozone-exposed groups, indicating the presence of a persistent inflammatory environment in the lung. Increased inflammation and Lm (mean linear intercept) scores were observed in chronic exposed mice, indicating emphysematous changes were present in lungs of chronic exposed mice. The antioxidative stress response was active (indicated by increased Nrf2 activity and protein) after 1 week of ozone exposure, but this ability was lost after 3 and 6 weeks of ozone exposure. The transcription factor HIF-1α was elevated in 3- and 6-week ozone-exposed mice and this was associated with increased gene expression levels of several HIF-1α target genes including Hdac2 (histone deacetylase 2), Vegf (vascular endothelial growth factor), Keap1 (kelch-like ECH-associated protein 1) and Mif (macrophage migration inhibitory factor). HDAC2 protein was found to be phosphorylated and carbonylated in nuclear and cytoplasm fractions, respectively, and was associated with a decrease in DNA-binding activity and protein expression of HDAC2. Decreased HDAC2 activity, most likely a direct result of protein modification, in combination with the loss of the antioxidative stress response and activation of the HIF-1α pathway, contribute to the inflammatory response and emphysema observed in ozone-exposed mice.

BACKGROUND

Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Earlier we reported maggot secretions to inhibit pro-inflammatory responses of human monocytes. The aim of this study was to investigate the effect of maggot secretions on the differentiation of monocytes into pro-inflammatory (MØ-1) and anti-inflammatory/pro-angiogenic macrophages (MØ-2) as these cells play a central role in wound healing.

RESULTS

Freshly isolated monocytes were incubated with secretions and GM-CSF or M-CSF for 6 days and then stimulated with LPS or LTA for 18 h. The expression of cell surface molecules and the levels of cytokines, chemokines and growth factors in supernatants were measured. Our results showed secretions to affect monocyte-macrophage differentiation leading to MØ-1 with a partial MØ-2-like morphology but lacking CD163, which is characteristic for MØ-2. In response to LPS or LTA, secretions-differentiated MØ-1 produced less pro-inflammatory cytokines (TNF-alpha, IL-12p40 and MIF) than control cells. Similar results were observed for MØ-2 when stimulated with low concentrations of LPS. Furthermore, secretions dose-dependently led to MØ-1 and MØ-2 characterized by an altered chemokine production. Secretions led to MØ-2, but not MØ-1, producing enhanced levels of the growth factors bFGF and VEGF, as compared to control cells. The expression of cell-surface receptors involved in LPS/LTA was enhanced by secretions, that of CD86 and HLA-DR down-regulated, while receptors involved in phagocytosis remained largely unaffected.

CONCLUSIONS

Maggot secretions skew the differentiation of monocytes into macrophages away from a pro-inflammatory to a pro-angiogenic type.

The presence of mass-independently fractionated sulphur isotopes (MIF-S) in many sedimentary rocks older than approximately 2.4 billion years (Gyr), and the absence of MIF-S in younger rocks, has been considered the best evidence for a dramatic change from an anoxic to oxic atmosphere around 2.4 Gyr ago. This is because the only mechanism known to produce MIF-S has been ultraviolet photolysis of volcanic sulphur dioxide gas in an oxygen-poor atmosphere. Here we report the absence of MIF-S throughout approximately 100-m sections of 2.76-Gyr-old lake sediments and 2.92-Gyr-old marine shales in the Pilbara Craton, Western Australia. We propose three possible interpretations of the MIF-S geologic record: (1) the level of atmospheric oxygen fluctuated greatly during the Archaean era; (2) the atmosphere has remained oxic since approximately 3.8 Gyr ago, and MIF-S in sedimentary rocks represents times and regions of violent volcanic eruptions that ejected large volumes of sulphur dioxide into the stratosphere; or (3) MIF-S in rocks was mostly created by non-photochemical reactions during sediment diagenesis, and thus is not linked to atmospheric chemistry.

Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T(4)) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T(4) concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T(4) (or its hormonally inert isomer D-T(4)) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T(4) significantly improved survival in mice with severe sepsis. To examine the specificity of the MIFT(4) interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T(4) or vehicle. D-T(4) significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T(4) may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T(4) as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T(4) concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T(4) may be beneficial in improving outcome from sepsis.

The macrophage migration-inhibitory factor (MIF) is a pro-inflammatory cytokine first known for its effect on macrophage migration and activation. Recent studies have shown that MIP plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of MIF on human chondrosarcoma cells are largely unknown. In the present study, MIF was found to increase the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by MIF treatment, and the MIF-induced expression of integrin and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. In addition, migration-prone sublines demonstrated that increased cell migration ability was correlated with increased expression of MIF and αvβ3 integrin. Taken together, our results indicate that MIF enhanced the migration of the chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/NF-κB signal transduction pathway.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been shown to play a role in wound-healing processes. In this study, we investigated whether protease-activated receptor (PAR)-1 and PAR-2 mediated MIF expression in human endothelial cells. Thrombin, factor Xa (FXa), and trypsin induced MIF expression in human dermal microvascular endothelial cells and human umbilical vein endothelial cells, but other proteases, including kallikrein and urokinase, failed to do so. Thrombin-induced MIF mRNA expression was significantly reduced by the thrombin-specific inhibitor hirudin. Thrombin receptor activation peptide-6, a synthetic PAR-1 peptide, induced MIF mRNA expression, suggesting that PAR-1 mediates MIF expression in response to thrombin. The effects of FXa were blocked by antithrombin III, but not by hirudin, indicating that FXa might enhance MIF production directly rather than via thrombin stimulation. The synthetic PAR-2 peptide SLIGRL-NH(2) induced MIF mRNA expression, showing that PAR-2 mediated MIF expression in response to FXa. Concerning the signal transduction, a mitogen-activated protein kinase kinase inhibitor (PD98089) and a nuclear factor (NF)-kappaB inhibitor (SN50) suppressed the up-regulation of MIF mRNA in response to thrombin, FXa, and PAR-2 agonist stimulation, whereas a p38 inhibitor (SB203580) had little effect. These facts indicate that up-regulation of MIF by thrombin or FXa is regulated by p44/p42 mitogen-activated protein kinase-dependent pathways and NF-kappaB-dependent pathways. Moreover, we found that PAR-1 and PAR-2 mRNA expression in endothelial cells was enhanced by MIF. Furthermore, we examined the inflammatory response induced by PAR-1 and PAR-2 agonists injected into the mouse footpad. As shown by footpad thickness, an indicator of inflammation, MIF-deficient mice (C57BL/6) were much less sensitive to either PAR-1 or PAR-2 agonists than wild-type mice. Taken together, these results suggest that MIF contributes to the inflammatory phase of the wound healing process in concert with thrombin and FXa via PAR-1 and PAR-2.

Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity, improved wound healing, and an increase in levels of arginase and other transcripts indicative of a resolution phase of wound healing. This study demonstrates the potential of MIF inhibition in SCI and the utility of nanocarrier-mediated drug delivery selectively to the injured cord.

This study investigates whether a set of ten potential breast cancer serum biomarkers and cancer antigens (osteopontin (OPN), haptoglobin, cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), prolactin, cancer antigen 19-9 (CA19-9), α-fetoprotein (AFP), leptin and migration inhibitory factor (MIF)) can predict early stage breast cancer in samples collected before clinical diagnosis (phase III samples). We performed a nested case-control study within the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort. We examined to what extent the biomarker panel could discriminate between 68 women diagnosed with breast cancer up to three years after enrollment and 68 matched healthy controls (all 56-64 years at baseline). Using a quantitative bead-based multiplexed assay, we determined protein concentrations in serum samples collected at enrollment. Principal Component Analysis (PCA) and Random Forest (RF) analysis revealed that on the basis of all ten proteins, early cases could not be separated from controls. When we combined serum protein concentrations and subject characteristics related to breast cancer risk in the RF analysis, this did not result in classification accuracy scores that could correctly classify the samples (sensitivity: 50%, specificity: 50%). Our findings indicate that this panel of selected tumor markers cannot be used for diagnosis of early breast cancer.

Sex is an important factor in the response to ischemic insults in both the laboratory and the clinic. Inflammation and cell death are points where sex-specific pathways diverge in stroke, and serum estrogen level status affect the response to inflammation. The cytokine macrophage migration inhibitory factor (MIF) is detrimental in experimental stroke models in male animals. However MIF is known to have sex-specific actions on inflammation and wound healing. The role of MIF in the ischemic female brain has not been evaluated. A transient middle cerebral artery occlusion (MCAO/90min) model was used to induce stroke in male, intact female, and ovariectomized female wildtype (WT) and MIF knockout (KO) mice. Infarct size was quantified 72h after stroke. Protein and cytokine levels were assessed post stroke. Female MIF KO mice had significantly larger strokes compared to WT females (mean hemispheric infarct±SEM: 63%±2% versus 29%±3%; n=8; p<0.05). Ovariectomized female MIF KO mice also had larger infarcts than ovariectomized WT littermates (70%±3% versus 47%±4%; n=11; p<0.05). In males, however, infarct size was equivalent between MIF KO and WT mice (63%±2% versus 67%±3%; n=9; p=0.25). There were no significant differences in cytokine levels at 6h post-infarct between mice of either genotype in brain. MIF KO females displayed more microglial activation (ionized calcium binding adaptor molecule 1 (Iba1) immunofluorescence) after stroke than did WT mice or MIF KO males. The larger infarcts in MIF KO females were associated with an early increase in mitochondrial localization of Jun activation domain-binding protein 1 (JAB1). Loss of MIF exacerbated injury in the female brain after experimental stroke, which was independent of changes in pro-inflammatory cytokine levels. This response is sex-specific, and is in part independent of physiological serum levels of estrogen.

Endometriosis, the ectopic development of endometrial tissue, is, particularly in peritoneal endometriosis, believed to result from tubal reflux of menstrual tissue. The release of cytokines and growth factors by refluxed endometrial cells in response to peritoneal inflammatory stimuli may enhance the capability of endometrial cells to implant and grow into the peritoneal host tissue. Herein we report that interleukin 1 (IL1), a major proinflammatory cytokine that is overproduced by endometriosis women-derived peritoneal macrophages and found in elevated concentrations in the peritoneal fluid of patients with endometriosis, stimulates the synthesis and the secretion of macrophage migration inhibitory factor (MIF) by human endometrial stromal cells. IL1B (0.1-100 ng/ml) exerted dose- and time-dependent effects of MIF protein secretion and mRNA synthesis, as shown by ELISA and reverse transcription-polymerase chain reaction, respectively. IL1B appeared to induce MIF gene transcription via the kappaB nuclear transcription factor (NFkappaB), as shown by electrophoretic mobility shift assay and Western blot analysis of IkappaB phosphorylation. Curcumin (10(-8) M), which is known for inhibiting NFkappaB activation, inhibited IL1B-induced MIF secretion as well as NFkappaB nuclear translocation and DNA binding. Taken together, these findings clearly show that IL1B up-regulates the expression of MIF in endometrial stromal cells in vitro and acts via NFkappaB. This may play an important role in the physiology of the human endometrium and the pathophysiology of endometriosis considering the immunomodulatory properties of MIF as well as its role in cell growth, angiogenesis and tissue remodeling.

BACKGROUND

Cardiovascular surgery with cardiopulmonary bypass (CPB) has improved in past decades, but inflammatory activation in this setting is still unpredictable and is associated with several postoperative complications. Perioperative levels of macrophage migration inhibitory factor (MIF) and other inflammatory mediators could be implicated in adverse outcomes in cardiac surgery.

METHODS

Serum levels of MIF, monocyte chemoattractant protein (MCP)-1, soluble CD40 ligand, IL-6 and IL-10 from 93 patients subjected to CPB were measured by enzyme-linked immunosorbent assay and compared with specific and global postoperative organ dysfunctions through multiple organ dysfunction score (MODS) and sequential organ failure assessment (SOFA).

RESULTS

Most of the cytokines measured had a peak of production between 3 and 6 hours after CPB, but maximum levels of MIF occurred earlier, at the cessation of CPB. Among specific organ dysfunctions, the most frequent was hematological, occurring in 82% of the patients. Circulatory impairment was observed in 73.1% of the patients, and 51% of these needed inotropics or vasopressors within the first 24 hours after surgery. The third most frequent dysfunction was pulmonary, occurring in 48.4% of the patients. Preoperative levels of MIF showed a relevant direct correlation with the intensity of global organ dysfunction measured by SOFA (rho = 0.46, p < 0.001) and MODS (rho = 0.50, p < 0.001) on the third day after surgery. MCP-1 production was associated with postoperative thrombocytopenia, and MIF was related to the use of a high dose of vasopressors in patients with cardiovascular impairment and also to lower values of the ratio of partial arterial oxygen tension (PaO2) to fraction of inspired oxygen (FiO2) registered in the first 24 hours after CPB.

CONCLUSIONS

Despite the multifactorial nature of specific or multiple organ dysfunctions, MIF should be explored as a predicting factor of organ dysfunction, or even as a potential therapeutic target in decreasing postoperative complications.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

BACKGROUND

Hypoxia plays an important role in vascular remodeling and directly affects vascular smooth muscle cells (VSMC) functions. Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor, and recent evidence suggests an important role of MIF in the progression of atherosclerosis and restenosis. However, the potential link between hypoxia and MIF in VSMC has not been investigated. The current study was designed to test whether hypoxia could regulate MIF expression in human VSMC. The effect of modulating MIF expression on hypoxia-induced VSMC proliferation and migration was also investigated at the same time.

RESULTS

Expression of MIF mRNA and protein was up-regulated as early as 2 hours in cultured human VSMCs after exposed to moderate hypoxia condition (3% O2). The up-regulation of MIF expression appears to be dependent on hypoxia-inducible transcription factor-1alpha(HIF-1alpha) since knockdown of HIF-1alpha inhibits the hypoxia induction of MIF gene and protein expression. The hypoxia induced expression of MIF was attenuated by antioxidant treatment as well as by inhibition of extracellular signal-regulated kinase (ERK). Under moderate hypoxia conditions (3% O2), both cell proliferation and cell migration were increased in VSMC cells. Blocking the MIF by specific small interference RNA to MIF (MIF-shRNA) resulted in the suppression of proliferation and migration of VSMCs.

CONCLUSIONS

Our results demonstrated that in VSMCs, hypoxia increased MIF gene expression and protein production. The hypoxia-induced HIF-1alpha activation, reactive oxygen species (ROS) generation and ERK activation might be involved in this response. Both MIF and HIF-1alpha mediated the hypoxia response of vascular smooth muscle cells, including cell migration and proliferation.

A series of phenolic hydrazones were synthesized and evaluated for their inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity. Compound 7 emerged as a potent inhibitor of MIF with an IC50 of 130 nM. Compound 7 dose-dependently suppressed TNFalpha secretion from lipopolysaccharide stimulated macrophages. The therapeutic importance of the MIF inhibition by 7 is demonstrated by the significant protection from the lethality of sepsis when administration of the compound was initiated in a clinically relevant time frame.

Macrophage migration inhibitory factor (MIF), the pro-inflammatory cytokine, first described in 1966, plays an essential role in both, innate and adaptive immune response. It has been implicated in tumour growth and angiogenesis and it exerts an antagonistic action against glucocorticoid immunosuppressive effect. Its perplexing enzymatic tautomerase activity has attracted considerable interest in the last decade. It has been suggested, that a multitude of autoimmune/inflammatory/neoplastic disease states might benefit from therapeutic measures, targeting MIF. Hence, small molecule inhibitors of MIF are relentlessly sought as potential anti-inflammatory (antitumour) agents, while a true in vivo substrate for MIF still remains unidentified. One of the first studied MIF inhibitor group was the D-dopachrome family, and its carboxyderivatives have shown good inhibitory effect, as well as the fluorosubstituted phenylpyruvic acid class. The substance ISO-1 of isoxazoline skeleton was the first small molecular inhibitor of MIF, not related to its known substrates. N-acetyl-p-benzoquinone, an acetaminophen metabolite and its synthetic derivatives exerted submicromolar IC(50) values. An acetylenic compound, the 2-oxo-4-phenyl-3-butynoate is a potent active-site-directed irreversible inhibitor of the phenyl pyruvate tautomerase activity of MIF. Some oxygen heterocycles, coumarines and chromenes, have also drawn attention as MIF inhibitors. The alpha,beta-unsaturated carbonyl compounds constitute a large novel class of MIF inhibitors. Several potent inhibitors were found among the cinnamic acid derivatives, thealpha,beta-unsaturated cyclic ketones, and the natural curcuminoids. Some other plant derived compounds were also studied. One of the latest developments in the field is the synthesis of AVP-13546, an exceptionally potent inhibitor. The structural pattern of MIF enzyme inhibitors exhibits wide variety; compounds having quite different molecular backbones belong to the MIF inhibitor family. In this paper, the separate classes of MIF inhibitors are discussed.

OBJECTIVE

To establish the relationship between plasma levels of thioredoxin (Trx) and macrophage migration inhibitory factor (MIF) in systemic inflammatory stress syndrome (SIRS)/sepsis.

METHODS

Enzyme-linked immunosorbent assay measurements of Trx, MIF, IL-6, -8, and -10 and enzyme-linked fluorescent assay determination of procalcitonin (PCT) in plasma from patients with SIRS/sepsis, neutropenic sepsis, healthy volunteers and pre-oesophagectomy patients.

RESULTS

Thioredoxin was significantly higher in SIRS/sepsis patients [101.3 ng ml(-1), interquartile range (IQR) 68.7-155.6, n = 32] compared with that in healthy controls (49.5 ng ml(-1), IQR 31.4-71.1, P < 0.001, n = 17) or pre-oesophagectomy patients (40.5 ng ml(-1), IQR 36.9-63.2, P < 0.01, n = 7), but was not raised in neutropenics (n = 5). MIF levels were also significantly higher in SIRS/sepsis patients (12.1 ng ml(-1), IQR 9.5-15.5, n = 35), but not in the neutropenic group, when compared with healthy controls (9.3 ng ml(-1), IQR 7.3-10.7, P < 0.01, n = 20). Trx levels correlated, positively, with MIF levels and APACHE II scores. Plasma levels of IL-6, -8 and -10 and PCT increased significantly in patients with SIRS/sepsis (P < 0.001) and with neutropenic sepsis, but did not correlate with Trx or MIF levels.

CONCLUSIONS

Plasma levels of Trx, MIF, IL-6, -8, -10 and PCT were raised in patients with SIRS/sepsis. Comparisons between mediators suggest a unique correlation of Trx with MIF. Moreover, Trx and MIF differed from cytokines and PCT in that levels were significantly lower in patients with neutropenia compared with the main SIRS/sepsis group. By contrast, IL-8 and PCT levels were significantly greater in the neutropenic patient group. The link between MIF and Trx highlighted in this study has implications for future investigations into the pathogenesis of SIRS/sepsis.

OBJECTIVE

Macrophage migration inhibitory factor (MIF) and CD74 emerge as important players in pathogenesis and angiogenesis of several types of malignant tumors. The purpose of this study was to evaluate the expression of MIF and CD74 in cervical squamous cell carcinoma and explore the potential roles they play in cervical tumor angiogenesis.

METHODS

Macrophage migration inhibitory factor and CD74 expression was assessed by immunohistochemistry in 209 cases with various degrees of cervical epithelial lesions, including 40 normal cervical epithelia, 43 mild cervical intraepithelial neoplasia 1 (CIN 1), 41 moderate-severe cervical intraepithelial neoplasia (CIN 2 to 3), and 85 cervical squamous cell carcinomas (SCCs). CD34 staining was used for counting microvessel density. Semiquantitative reverse transcription polymerase chain reaction and Western blot were used to detect messenger RNA and protein levels of MIF and CD74 in normal and malignant cervical tissues and cervical cancer cell lines SiHa and C-33A. The concentration of vascular endothelial growth factor (VEGF) in the conditioned media of cervical cancer cells was analyzed by enzyme-linked immunosorbent assay.

RESULTS

Immunohistochemical analysis showed that MIF and CD74 expression was significantly higher in CIN than in the normal samples and higher in SCC than in CIN. The overexpression of MIF was correlated with deep stromal infiltration but not with the other clinicopathologic features of SCC. Correlation analyses revealed that MIF was positively related to CD74, and both protein levels were associated with microvessel density. Exogenous MIF induced VEGF secretion in SiHa and C-33A cells in a dose-dependent manner, which can be inhibited by MIF-specific inhibitor (ISO-1) or anti-CD74 antibody.

CONCLUSIONS

Overexpression of MIF and CD74 in SCC and its precancerous lesions and the up-regulation of VEGF secretion in cervical cancer cells indicate that MIF and CD74 may play critical roles in the pathogenesis and angiogenesis of cervical cancer.

Temozolomide (TMZ) is the most effective chemotherapeutic agent for glioblastoma (GBM). Resistance to this methylating agent is linked to DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). However, in recent studies MGMT status was not completely accurate as a predictor of TMZ response in GBM, suggesting other mechanisms of resistance. As part of an effort aimed at discovery of genes involved in TMZ resistance in GBM, the expression of CD74 was evaluated in GBM patient samples and the influence of CD74 on TMZ response was evaluated in GBM tumor models. Reverse transcription-polymerase-chain reaction (RT-PCR) demonstrated differential expression of CD74 mRNA among the GBM xenografts; 8 of 20 (40%) expressed CD74 mRNA. In a preliminary evaluation of whether CD74 expression might influence TMZ response, CD74 mRNA expression levels were inversely associated with in vivo TMZ resistance in 20 GBM xenograft lines (median survival 122 vs. 62.5 days; r = -0.48, P = 0.032). In follow up to this observation, CD74 shRNA knock down in U87 cells significantly suppressed in vitro proliferation and increased TMZ sensitivity as compared to a non-specific control shRNA. Consistent with an effect on proliferation and survival, silencing of CD74 by shRNA was associated with reduced Akt and Erk1/2 activation in response to stimulation by CD74 ligand macrophage-migration inhibition factor (MIF). Lastly, expression of CD74 protein was assessed in patient samples [nine anaplastic astrocytoma (AA), and 62 GBM] by immunohistochemistry, and appreciable expression was observed in 28% of samples. Collectively, these findings suggest that CD74 is expressed in a subset of high grade gliomas and may contribute to TMZ resistance.

Cancer cachexia induces loss of fat mass that accounts for a large part of the dramatic weight loss observed both in humans and in animal models; however, the literature does not provide consistent information regarding the set point of weight loss and how the different visceral adipose tissue depots contribute to this symptom. To evaluate that, 8-week-old male Wistar rats were subcutaneously inoculated with 1 ml (2×10(7)) of tumour cells (Walker 256). Samples of different visceral white adipose tissue (WAT) depots were collected at days 0, 4, 7 and 14 and stored at -80 °C (seven to ten animals/each day per group). Mesenteric and retroperitoneal depot mass was decreased to the greatest extent on day 14 compared with day 0. Gene and protein expression of PPARγ2 (PPARG) fell significantly following tumour implantation in all three adipose tissue depots while C/EBPα (CEBPA) and SREBP-1c (SREBF1) expression decreased over time only in epididymal and retroperitoneal depots. Decreased adipogenic gene expression and morphological disruption of visceral WAT are further supported by the dramatic reduction in mRNA and protein levels of perilipin. Classical markers of inflammation and macrophage infiltration (f4/80, CD68 and MIF-1α) in WAT were significantly increased in the later stage of cachexia (although showing a incremental pattern along the course of cachexia) and presented a depot-specific regulation. These results indicate that impairment in the lipid-storing function of adipose tissue occurs at different times and that the mesenteric adipose tissue is more resistant to the 'fat-reducing effect' than the other visceral depots during cancer cachexia progression.

Peptides can be transported across the blood-brain barrier by saturable transport systems. One system, characterized with radioactively labeled Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide), is specific for some of the small peptides with an N-terminal tyrosine, including Tyr-MIF-1, the enkephalins, beta-casomorphin, and dynorphin (1-8). Another separate system transports vasopressin-like peptides. The choroid plexus has at least one system distinguishable from those above that is capable of uptake and possibly transport of opiate-like peptides. The possibility of saturable transport of other peptides has been investigated to a varying degree. Specificity, stereo-specificity, saturability, allosteric regulation, modulation by physiologic and pharmacologic manipulations, and noncompetitive inhibition have been demonstrated to occur in peptide transport systems and suggest a role for them in physiology and disease.