D-dopachrome tautomerase (D-DT) is a newly described cytokine and a member of the macrophage migration inhibitory factor (MIF) protein superfamily. MIF is a broadly expressed pro-inflammatory cytokine that regulates both the innate and the adaptive immune response. MIF activates the MAP kinase cascade, modulates cell migration, and counter-acts the immunosuppressive effects of glucocorticoids. For many cell types, MIF also acts as an important survival or anti-apoptotic factor. Circulating MIF levels are elevated in the serum in different infectious and autoimmune diseases, and neutralization of the MIF protein via antibodies or small molecule antagonists improves the outcome in numerous animal models of human disease. Recently, a detailed investigation of the biological role of the closely homologous protein D-DT, which is encoded by a gene adjacent to MIF, revealed an overlapping functional spectrum with MIF. The D-DT protein also is present in most tissues and circulates in serum at similar concentrations as MIF. D-DT binds the MIF cell surface receptor complex, CD74/CD44, with high affinity and induces similar cell signaling and effector functions. Furthermore, an analysis of the signaling properties of the two proteins showed that they work cooperatively, and that neutralization of D-DT in vivo significantly decreases inflammation. In this review, we highlight the similarities and differences between MIF and D-DT, which we propose to designate "MIF-2", and discuss the implication of D-DT/MIF-2 expression for MIF-based therapies.

Macrophage migration inhibitory factor (MIF) is a multifunctional protein that exhibits an intrinsic thiol protein oxidoreductase activity and proinflammatory activities. In the present study to examine intracellular MIF redox function, exposure of MIF-deficient cardiac fibroblasts to oxidizing conditions resulted in a 2.3-fold increase (p < 0.001) in intracellular ROS that could be significantly reduced by adenoviral-mediated reexpression of recombinant MIF. In an animal model of myocardial injury by ischemia/reperfusion (I/R), MIF-deficient hearts exhibited higher levels of oxidative stress than did wild-type hearts, as measured by significantly higher oxidized glutathione levels (decreased GSH/GSSG ratio), increased protein oxidation, reduced aconitase activity, and increased mitochondrial injury (increased cytochrome c release). The increased myocardial oxidative stress after I/R was reflected by larger infarct size (INF) in MIF-deficient hearts versus wild-type (WT) hearts (21 ± 6% vs. 8 ± 3% INF/LV; p < 0.05). In vivo hemodynamic measurements showed that left ventricular (LV) contractile function of MIF-deficient hearts subjected to 15-min ischemia failed to recover during reperfusion compared with WT hearts (LV developed pressure and ± dP/dt; p = 0.02). These data represent the first in vivo evidence in support of a cardioprotective role of MIF in the postischemic heart by reducing oxidative stress.

BACKGROUND

T-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. Significantly, a comparative genome-scale transcriptional analysis of two T-cell subsets (CD4+ and CD8+) against each other and against the naturally mixed population (CD3+ cells) remains unexplored.

RESULTS

Comparison of the microarray-based gene expression patterns between CD3+ T cells, and the CD4+ and CD8+ subsets revealed largely conserved, but not identical, transcriptional patterns. We employed a Gene-Ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel T-cell activation genes and cell-type-specific genes associated with the immune response. We identified potential genes involved in the communication between the two subsets (including IL23A, NR4A2, CD83, PSMB2, -8, MIF, IFI16, TNFAIP1, POU2AF1, and OTUB1) and would-be effector-function-specific genes (XCL2, SLAMF7, TNFSF4, -5, -9, CSF3, CD48 and CD244). Chemokines induced during T-cell activation, but not previously identified in T cells, include CCL20, CXCL9, -10, -11 (in all three populations), and XCL2 (preferentially in CD8+ T cells). Increased expression of other unexpected cytokines (GPI, OSM and MIF) suggests their involvement in T-cell activation with their functions yet to be examined. Differential expression of many receptors, not previously reported in the context of T-cell activation, including CCR5, CCR7, IL1R2, IL1RAP, IL6R, TNFRSF25 and TNFRSF1A, suggests their role in this immune process. Several receptors involved in TCR activation (CD3D, CD3G, TRAT1, ITGAL, ITGB1, ITGB2, CD8A and B (CD8+ T-cell specific) along with LCK, ZAP70 and TYROBP were synchronously downregulated. Members of cell-surface receptors (HLA-Ds and KLRs), none previously identified in the context of T-cell activation, were also downregulated.

CONCLUSIONS

This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ populations made possible the identification of many immune-response genes not previously identified in the context of T-cell activation. Significantly, it made possible to identify the temporal patterns of many previously known T-cell activation genes, and also identify genes implicated in effector functions of and communication between CD4+ and CD8+ T cells.

OBJECTIVE

The hyperplasia of fibroblast-like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation.

METHODS

Human RA FLS were treated with recombinant MIF, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and/or anti-MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor kappa B (NF-kappa B) and mitogen-activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively.

RESULTS

FLS proliferation was significantly increased by MIF. IL-1 beta and TNFalpha also induced proliferation, but these effects were prevented by neutralization with anti-MIF mAb. Activation of NF-kappa B was induced by IL-1 beta, but not by MIF. Anti-MIF mAb had no effect on IL-1 beta-induced NF-kappa B nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal-regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF-kappa B antagonism, prevented the effect of MIF on FLS proliferation.

CONCLUSIONS

These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL-1 beta and TNFalpha. In addition, the effects of MIF on FLS activation are independent of NF-kappa B, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.

Traumatic brain injury (TBI) initiates a cascade of cellular and molecular responses including both pro- and anti-inflammatory. Although post-traumatic hypothermia has been shown to improve outcome in various models of brain injury, the underlying mechanisms responsible for these effects have not been clarified. In this study, inflammation cDNA arrays and semi-quantitative RT-PCR were used to detect genes that are differentially regulated after TBI. In addition, the effect of post-traumatic hypothermia on the expression of selective genes was also studied. Rats (n = 6-8 per group) underwent moderate fluid-percussion (F-P) brain injury with and without hypothermic treatment (33 degrees C/3 h). RNA from 3-h or 24-h survival was analyzed for the expression of IL1-beta, IL2, IL6, TGF-beta2, growth-regulated oncogene (GRO), migration inhibitory factor (MIF), and MCP (a transcription factor). The interleukins IL-1beta, IL-2, and IL-6 and TGF-beta and GRO were strongly upregulated early and transiently from 2- to 30-fold over sham at 3 h, with normalization by 24 h. In contrast, the expressions of MIF and MCP were both reduced by TBI compared to sham. Post-traumatic hypothermia had no significant effect on the acute expression of the majority of genes investigated. However, the expression of TGF-beta2 at 24 h was significantly reduced by temperature manipulation. The mechanism by which post-traumatic hypothermia is protective may not involve a general genetic response of the inflammatory genes. However, specific genes, including TGF-beta2, may be altered and effect cell death mechanisms after TBI. Hypothermia differentially regulates certain genes and may target more delayed responses underlying the secondary damage following TBI.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pivotal role in several immunoinflammatory and autoimmune diseases. In this study we examined the role of MIF in the development of immunoinflammatory diabetes induced in susceptible strains of mice by multiple low doses of streptozotocin. We found that MIF protein was significantly elevated in islet cells during the development of diabetes, and that targeting MIF activity with either neutralizing antibody or the pharmacological inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, markedly reduced clinical and histopathological features of the disease, such as hyperglycemia and insulitis. Lymphocytes from mice treated with the MIF inhibitors exhibited reduction of both islet antigen-specific proliferative responses and adhesive cell-cell interactions. Neutralization of MIF also down-regulated the ex vivo secretion of the proinflammatory mediators, TNF-alpha, interferon-gamma, and nitric oxide, while augmenting that of the antiinflammatory cytokine, IL-10. This study provides the first in vivo evidence for a critical role for MIF in the immune-mediated beta-cell destruction in an animal model of human type 1 diabetes mellitus and identifies a new therapeutic strategy for the prevention and treatment of this disease in humans that is based on the selective inhibition of MIF activity.

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.

BACKGROUND

Macrophage migration inhibitory factor (MIF), a cytokine that controls cell-mediated inflammatory responses, is upregulated in atherogenesis; however, its functional contribution to lesion development has not been evaluated.

RESULTS

We studied the role of MIF on neointima lesion formation after wire-induced injury of carotid arteries in apolipoprotein E-deficient (apoE(-/-)) mice. Immunohistochemistry revealed that MIF expression was detectable in endothelial cells before injury and upregulated in smooth muscle cells (SMCs) 24 hours after endothelial denudation. Three weeks after injury, MIF was predominantly found in endothelial cells and macrophage-derived foam cells. Neutralizing MIF with a monoclonal antibody resulted in a marked reduction of neointimal macrophages and inhibited transformation of macrophages into foam cells. Conversely, the content of SMCs and of collagen in the neointima were increased, amounting to a slight but not significant reduction in neointima and media size after 3 weeks of MIF monoclonal antibody treatment. Notably, serum levels of the cytokines IL-2, IL-4, IL-6, IL-10, and tumor necrosis factor were increased in MIF monoclonal antibody-treated mice. In vitro flow assays revealed that MIF pretreatment of aortic endothelium enhanced monocyte recruitment and that the monocyte arrest induced by oxidized LDL is mediated by endothelial MIF, as shown by monoclonal antibody inhibition.

CONCLUSIONS

Inhibition of MIF resulted in a shift in the cellular composition of neointimal plaques toward a stabilized phenotype with reduced macrophage/foam cell content and increased SMC content. This might be attributable to a reduction of monocyte recruitment mediated by endothelial MIF.

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Food-borne trematodiasis is an emerging public health problem, including in Lao PDR. We investigated the diversity of intestinal helminthes and polyparasitism in patients with hepatobiliary or intestinal symptoms in hospital and community-based surveys. Stool samples from 232 individuals aged>>or=15 years were examined by the Kato-Katz method (three samples) and a formalin ethyl-acetate concentration technique (one sample). Opisthorchis viverrini and minute intestinal flukes (MIF) were common, with prevalences of 86.2% and 62.9%, respectively. Hookworm was the predominant soil-transmitted helminth (65.9%). The prevalences of Taenia spp., Strongyloides stercoralis and Trichuris trichiura were 22.8%, 10.3% and 8.6%, respectively. Additionally, 97 individuals were purged; O. viverrini and Haplorchis taichui were found in 95 and 76 participants, respectively. Other trematodes included Phaneropsolus bonnei (22.7%), Prosthodendrium molenkampi (14.4%), Haplorchis pumilio (5.2%), Haplorchis yokogawai (3.1%) and Echinochasmus japonicus (3.1%). Co-infection with O. viverrini and MIFs was rampant (81.4%). Polytrematode infection is highly prevalent in Lao PDR and hence requires urgent attention.

Microglia are observed in the early developing forebrain and contribute to the regulation of neurogenesis through still unravelled mechanisms. In the developing cerebral cortex, microglia cluster in the ventricular/subventricular zone (VZ/SVZ), a region containing Cxcl12-expressing basal progenitors (BPs). Here we show that the ablation of BP as well as genetic loss of Cxcl12 affect microglia recruitment into the SVZ. Ectopic Cxcl12 expression or pharmacological blockage of CxcR4 further supports that Cxcl12/CxcR4 signalling is involved in microglial recruitment during cortical development. Furthermore, we found that cell death in the developing forebrain triggers microglial proliferation and that this is mediated by the release of macrophage migration inhibitory factor (MIF). Finally, we show that the depletion of microglia in mice lacking receptor for colony-stimulating factor-1 (Csf-1R) reduces BPs into the cerebral cortex.

OBJECTIVE

Macrophage migration inhibitory factor (MIF) is implicated in tumorigenesis. This study was conducted to determine whether MIF expression is associated with gastric pathology and whether MIF expression is increased in malignant gastric cells in vitro.

METHODS

Patients with a normal gastric mucosa, Helicobacter pylori infected gastritis, intestinal metaplasia, and gastric adenocarcinoma were included. Immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used to determine MIF expression in gastric epithelial cells and MIF levels in serum, respectively. Five gastric cancer cell lines (AGS, MKN-45, MKN-28, MGC-803, and SGC-7901) and one non-malignant gastric cell line (GES-1) were cultured for 24 hours. MIF protein in the supernatant and MIF mRNA in cultured cells were measured by ELISA and reverse transcription-polymerase chain reaction, respectively.

RESULTS

The percentage of MIF expressing epithelial cells was low in normal mucosa (12%) but substantially higher in gastritis (52%), intestinal metaplasia (66%), and gastric cancer (96%) (p<0.001, ANOVA). Serum MIF levels were low in patients with a normal mucosa (576 (82) pg/ml) but higher in patients with gastritis (2100 (349) pg/ml), intestinal metaplasia (4498 (253) pg/ml), and gastric cancer (9737 (1249) pg/ml) (p<0.001, ANOVA). There was a correlation between epithelial MIF expression and serum MIF levels (r = 0.776, p<0.001). In vitro, expression of MIF protein and mRNA was increased in malignant cells compared with non-malignant cells.

CONCLUSIONS

Epithelial and serum MIF expression was progressively increased in H pylori induced gastritis, intestinal metaplasia, and gastric cancer, suggesting that MIF is involved in gastric carcinogenesis and may be a valuable biomarker for the early detection of gastric cancer.

Mesenchymal stromal cells (MSCs) are inherently tumor homing and can be isolated, expanded, and transduced, making them viable candidates for cell therapy. This tumor tropism has been used to deliver anticancer therapies to various tumor models. In this study, we sought to discover which molecules are the key effectors of human MSC tumor homing in vitro and using an in vivo murine model. In this study, we discover a novel role for macrophage migration inhibitory factor (MIF) as the key director of MSC migration and infiltration toward tumor cells. We have shown this major role for MIF using in vitro migration and invasion assays, in presence of different receptor inhibitors and achieving a drastic decrease in both processes using MIF inhibitor. Additionally, we demonstrate physical interaction between MIF and three receptors: CXCR2, CXCR4, and CD74. CXCR4 is the dominant receptor used by MIF in the homing tumor context, although some signaling is observed through CXCR2. We demonstrate downstream activation of the MAPK pathway necessary for tumor homing. Importantly, we show that knockdown of either CXCR4 or MIF abrogates MSC homing to tumors in an in vivo pulmonary metastasis model, confirming the in vitro two-dimensional and three-dimensional assays. This improved understanding of MSC tumor tropism will further enable development of novel cellular therapies for cancers.

Atherosclerosis is an inflammatory response of the arterial wall to "injury", which is prominently driven by cytokines. The inflammatory mediator macrophage migration inhibitory factor (MIF) is a unique cytokine that was recently associated with atherogenesis. Here, we have investigated whether MIF has a role in spontaneous atherosclerosis by studying apolipoprotein E-deficient (ApoE(-/-)) mice treated with neutralizing anti-MIF monoclonal antibody and comparison with isotype IgG-treated controls. After 14 weeks, the aortas and heart valves were analyzed for inflammatory status, macrophage content and plaque areas. MIF expression in the aortic wall was elevated upon spontaneous atherogenesis, with foam cells representing a major source. Of note, MIF blockade led to a marked reduction in intimal Mac-1-positive macrophages. Similarly, treatment with anti-MIF antibody led to a reduction of a variety of inflammatory mediators typically associated with atherosclerosis including the circulating levels of fibrinogen, MIF and IL-6. Importantly, the local aortic expression of ICAM-1, MMP-2, TNF, IL-12, and CD40L was reduced by MIF blockade, as were the levels of the phospho-c-Jun and C/EBPbeta transcription factors. The observed strong reduction of inflammatory parameters by anti-MIF treatment was associated with a small, yet non-significant, reduction in aortic plaque area. Thus, although MIF's role is not directly linked to plaque volume expansion, in this mouse model of spontaneous atherogenesis, MIF plays an important role in intimal inflammation.

Retinal microglial cells may have a role in both degeneration and neuroprotection of retinal ganglion cells (RGC) after optic nerve (ON) section. We have used NDPase enzymohistochemistry to label adult rat retinal microglial cells and have studied these cells under normal conditions, after left ON section, and after left ON section and eye puncture or intravitreal injection of different substances: vehicle, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), or macrophage inhibitory factor (MIF). Resident microglial cells are present in four layers in the adult rat retina: the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Left ON section induces microglial activation in the ipsilateral and contralateral retina as manifested by stronger staining intensity in both retinas and increased microglial cell densities in the NFL, IPL, and GCL of the ipsilateral retina. Left ON section followed by left eye puncture or intravitreal injection increases microglial cell density in both retinas and induces changes in the microglial cells of the ipsilateral retina that vary depending on the substance injected: BDNF injections delay microglial activation, possibly through retinal ganglion cell neuroprotection, whereas NT3 partially inhibits microglial activation in the NFL; MIF injections have no clear effects on microglial activation. In conclusion, retinal microglial cells become activated after an ON section and react more intensely when the eye is also punctured or injected, and this response may be altered by using neurotrophic factors, although the effects of MIF are less clear.

Originally discovered and named as an in vitro inhibitor of macrophage migration, the cytokine macrophage migration inhibitory factor (MIF) has now been shown to be a key regulator of acute and chronic immuno-inflammatory conditions including rheumatoid arthritis (RA), atherosclerosis, and more recently systemic lupus erythematosus (SLE). Common inflammatory events in these diseases include activation of cells and infiltration by immune cells at the site of injury. MIF actively participates in multiple stages of the inflammatory response, acting on cells directly and/or potentiating the effects entrained by other stimuli. The overlap of inflammatory processes operating in these diseases, the known activities of MIF, and the observation of atherosclerosis as a major comorbidity of RA and SLE, make MIF a strong candidate for therapeutic targeting in these diseases. Moreover, the unique relationship between MIF and glucocorticoids, commonly used in the treatment of RA and SLE but associated with significant side effects, highlights the potential of MIF as a 'steroid sparing' therapeutic target encompassing all three conditions.

Macrophage migration inhibitory factor (MIF) may contribute to multiple aspects of tumor progression, including control of cell proliferation, differentiation, cell survival and angiogenesis. However, the potential roles of MIF in regulating hepatocellular carcinoma (HCC) tumor cell migration and the expression of angiogenic factors by HCC tumor cells have not been studied yet. In our study, we reported that intracellular MIF mRNA and protein were overexpressed in HCC tissues compared to nontumor tissues by using in situ hybridization and immunohistochemic staining. HCC tumor cell lines also secreted large amounts of MIF into the supernatants of tumor cell culture. To assess the role of MIF in HCC, we employed the transwell invasion chamber to study the effect of MIF on tumor cell migration. Our results showed that recombinant MIF and the supernatants of tumor cell line culture could enhance the invasion and migration of HCC cells. This effect can be inhibited by the addition of a neutralizing anti-MIF antibody. We observed that increased MIF serum levels correlated with higher levels of interleukin-8 (IL-8) in the sera of patients with HCC than in normal volunteers. We therefore hypothesized that MIF may regulate the production of angiogenic factors by HCC cells. To test this hypothesis, we examined the effect of MIF treatment on vascular endothelial growth factor (VEGF) and IL-8 expression by HCC cell lines. MIF induced a significant dose-dependent increase in IL-8 and VEGF production. Taken together, our results indicated that MIF may act as an autocrine-acting factor that stimulates angiogenesis and metastasis in HCC by promoting expression of angiogenic factors and migration of tumor cells. A more detailed understanding of the MIF regulatory mechanisms involved may provide insight into new direction in the treatment of HCC.

BACKGROUND

We have recently demonstrated that macrophage migration inhibitory factor (MIF) plays a pathogenic role in experimental glomerulonephritis (GN). The aim of the current study was to investigate MIF expression in human GN.

METHODS

MIF expression was examined by in situ hybridization and immunohistochemistry staining in 65 biopsies from a variety of glomerulonephridities.

RESULTS

There is constitutive expression of MIF mRNA and protein in normal human kidney that is largely restricted to tubular epithelial cells and to some glomerular epithelial cells. There was little change in the pattern of MIF expression in nonproliferative forms of GN such as minimal change disease and membranous GN. However, there was a marked increase in both glomerular and tubular MIF expression in proliferative forms of GN, including focal segmental glomerulosclerosis (FGS), lupus nephritis, crescentic GN, and mesangiocapillary proliferative GN. The prominent macrophage and T-cell infiltrate in these diseases were largely restricted to areas with marked up-regulation of MIF expression, contributing to glomerular hypercellularity, glomerular focal segmental lesions, crescent formation, tubulitis, and granulomatous lesions. De novo MIF expression was evident in glomerular endothelial cells and mesangial cells in proliferative forms of GN. In addition, many infiltrating macrophages and T cells showed MIF mRNA and protein expression. Quantitative analysis found that increased glomerular and tubular MIF expression gave a highly significant correlation with macrophage and T-cell accumulation, the severity of histologic lesions, and the loss of creatinine clearance.

CONCLUSIONS

Renal MIF expression is markedly up-regulated in proliferative forms of human GN, and this correlates with leukocyte infiltration, histologic damage, and renal function impairment. These results suggest that MIF may be an important mediator of renal injury in progressive forms of human GN. Based on these findings, together with the known pathogenic role of MIF in experimental GN, we propose that MIF is an attractive therapeutic target in the treatment of progressive forms of GN.

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms (single-nucleotide polymorphisms (SNPs)) (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3' end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.

BACKGROUND

Macrophage migration inhibitory factor (MIF) was one of the first lymphokine activities to be discovered and was described almost 30 years ago to be a soluble factor(s) produced by activated T lymphocytes. In more recent studies, MIF has been "rediscovered" to be an abundant, pre-formed constituent of the anterior pituitary gland and the macrophage, and to be a critical component in the host response to septic shock. Pituitary-derived MIF enters the circulation after infectious or stressful stimuli and appears to act to counterregulate glucocorticoid suppression of cytokine production.

METHODS

Immunoelectron microscopy utilizing a combination of anti-MIF and anti-pituitary hormone-specific antibodies was used to study the ultrastructural localization of MIF within the anterior pituitary gland. Pituitaries were obtained from resting, unstimulated mice and from mice 16 hr after endotoxin administration. The release of MIF also was investigated in vitro by examining the effect of corticotropin-releasing hormone (CRH_ on the AtT-20, corticotrophic cell line.

RESULTS

MIF localizes to granules present exclusively in ACTH and TSH secreting cells. Within each cell type, a subset of granules was found to contain both MIF and ACTH, or MIF and TSH. The pituitary content of MIF-containing granules decreased significantly after experimentally induced endotoxemia. In seven pituitaries examined 16 hr after LPS injection, the number of MIF-positive granules diminished by 38% in corticotrophic cells and by 48% in thyrotrophic cells when compared with controls (p < 0.05). CRH was observed to be a potent MIF secretagogue in vitro, inducing the release of MIF from corticotrophic cells at concentrations lower than that required for ACTH release.

CONCLUSIONS

These data provide ultrastructural information that identify MIF to be a novel anterior pituitary hormone, support earlier studies showing a time-dependent release of pituitary MIF during endotoxemia, and suggest an important, systemic role for MIF in the stress response to infection and other stimuli.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a pivotal cytokine that regulates inflammatory and immune responses. Recently, many investigators reported that MIF is expressed highly in several tumors, including hepatocellular carcinoma (HCC). However, the role of MIF in tumor angiogenesis and patient prognosis has not been examined in patients with HCC.

METHODS

The authors evaluated MIF expression in 56 samples of HCC by Western blot analysis, and the results were correlated with clinicopathologic factors and patient prognosis. MIF localization was determined by immunohistochemical methods, and the results were compared with tumor microvessel density (MVD), as assessed by anti-CD34 antibody. Furthermore, to validate the role of MIF in angiogenesis, both MIF expression during culture of HCC cells (using the Hep3B, HepG2, and Huh7 cell lines) under hypoxic condition and the angiogenic potential of recombinant MIF in an in vitro angiogenic model were examined.

RESULTS

Tumors with high MIF expression had high alpha-fetoprotein levels (P = 0.049) and frequent intrahepatic recurrence (P = 0.043). Immunohistochemical MIF scores had a significant correlation with MVD (P = 0.007). Patients who had tumors with high MIF expression levels had a significantly worse (P = 0.025) disease-free survival, and this finding remained significant as an independent prognostic factor in the multivariate analysis. Hep3B cells had high expression of MIF at 6 hours and 12 hours after hypoxic stress and exogenous MIF stimulated endothelial tube formation in in vitro angiogenesis.

CONCLUSIONS

The current findings suggest that MIF expression may play a pivotal role in the dismal prognosis of patients with HCC that may be attributable to the modulation of angiogenesis.

BACKGROUND

Myelin Oligodendrocyte Glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model for multiple sclerosis (MS). During the of progression of EAE, microglia, the immunocompetent cells of the brain, become activated and accumulate around demyelinated lesions. Microglial activation is mediated by the extracellular protease tissue Plasminogen Activator (tPA), and mice lacking tPA display altered EAE progression. In this study, we have used pharmacological inhibitors and stimulators of microglial/macrophage activation to examine the temporal requirement for microglial activation in EAE progression and to determine whether such approaches might potentially be of therapeutic value.

RESULTS

Intervention using the tripeptide macrophage/microglia inhibitory factor MIF (TKP) and the tetrapeptide macrophage/microglial stimulator tuftsin (TKPR) attenuated EAE symptoms and revealed that the timing of macrophage/microglial activation is critical for the clinical outcome of EAE. We show that the disease progression can potentially be manipulated favorably at early stages by altering the timing of microglial activation, which in turn alters the systemic immune response to favor upregulation of T helper cell 2 genes that promote recovery from EAE.

CONCLUSIONS

Preventative and therapeutic modulation of macrophage/microglial activity significantly alters the outcome of EAE at symptomatic stages. Specific molecular targets have been identified that represent potential avenues of exploration for the treatment and prevention of MS.

BACKGROUND

Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently used markers.

METHODS

We used a combination of in-house and commercially available multiplex immunoassays based on Luminex xMAP technology to assay biomarkers of potential interest in EDTA-plasma samples.

RESULTS

A 3-plex assay for soluble urokinase plasminogen activator receptor (suPAR), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha human cytokine panel. No cross-reactivity was observed when the assays were combined. Correlation between values obtained with the 8-plex, the 5-cytokine panel, the 3 in-house 1-plex assays, and a suPAR ELISA ranged from 0.86 to 0.99. Mean within- and between-run CVs were 8.0% and 11%, respectively. Recoveries of suPAR, sTREM-1, and MIF calibrators were 108%, 88%, and 51%, respectively. In plasma collected from 10 patients with bacterial sepsis confirmed by blood culture, the assay detected significantly increased concentrations of all 8 analytes compared with healthy controls.

CONCLUSIONS

A commercially available xMAP panel can be expanded with markers of interest. The combined multiplex assay can measure the 8 analytes with high reproducibility. The xMAP technology is an appealing tool for assaying conventional cytokines in combination with new markers.

Metabolism of xenobiotics remains a central challenge for the discovery and development of drugs, cosmetics, nutritional supplements, and agrochemicals. Metabolic transformations are frequently related to the incidence of toxic effects that may result from the emergence of reactive species, the systemic accumulation of metabolites, or by induction of metabolic pathways. Experimental investigation of the metabolism of small organic molecules is particularly resource demanding; hence, computational methods are of considerable interest to complement experimental approaches. This review provides a broad overview of structure- and ligand-based computational methods for the prediction of xenobiotic metabolism. Current computational approaches to address xenobiotic metabolism are discussed from three major perspectives: (i) prediction of sites of metabolism (SOMs), (ii) elucidation of potential metabolites and their chemical structures, and (iii) prediction of direct and indirect effects of xenobiotics on metabolizing enzymes, where the focus is on the cytochrome P450 (CYP) superfamily of enzymes, the cardinal xenobiotics metabolizing enzymes. For each of these domains, a variety of approaches and their applications are systematically reviewed, including expert systems, data mining approaches, quantitative structure-activity relationships (QSARs), and machine learning-based methods, pharmacophore-based algorithms, shape-focused techniques, molecular interaction fields (MIFs), reactivity-focused techniques, protein-ligand docking, molecular dynamics (MD) simulations, and combinations of methods. Predictive metabolism is a developing area, and there is still enormous potential for improvement. However, it is clear that the combination of rapidly increasing amounts of available ligand- and structure-related experimental data (in particular, quantitative data) with novel and diverse simulation and modeling approaches is accelerating the development of effective tools for prediction of in vivo metabolism, which is reflected by the diverse and comprehensive data sources and methods for metabolism prediction reviewed here. This review attempts to survey the range and scope of computational methods applied to metabolism prediction and also to compare and contrast their applicability and performance.