MIF is an important regulator of innate and adaptive immunity, which is produced by a variety of cell types including activated T cells and macrophages. We examined the effects of MIF on osteoclastogenesis in bone marrow (BM) cultures from WT and MIF-deficient (KO) mice as well as the bone mass of MIF KO mice. Exogenous MIF inhibited osteoclast formation in BM cultures by decreasing fusion in cells that were treated with M-CSF and RANKL. However, inhibition of OCL formation by MIF treatment was not mediated by fusion-related molecules in heterogeneous bone marrow cultures. BM cultures from MIF KO mice that were treated with M-CSF and RANKL, PTH or vitamin D had significantly increased OCL number compared to cells from WT mice. MIF also significantly inhibited OCL formation in cultures of RAW 264.7 cells that were treated with RANKL. In addition, the number of CFU-GM and Mac-1(+) cells in the BM of MIF KO mice was greater than in WT controls. Trabecular bone volume (TBV) in the femurs and vertebrae of MIF KO mice was decreased compared to WT mice. In addition, serum bone resorption and formation markers were decreased in MIF KO mice compared to WT mice. These results demonstrate that MIF has inhibitory effects on OCL formation in vitro. We also found that BM cell cultures from MIF KO mice had an increased capacity to form osteoclasts. Furthermore, MIF KO animals had significantly decreased TBV with low turnover. We conclude that MIF is an inhibitor of osteoclastogenesis in vitro, which may regulate bone turnover via indirect mechanism in vivo.

Macrophage migration inhibitory factor (MIF) from vertebrate species is a molecule that exerts a wide-range of effects in inflammatory responses, cell activation and cell differentiation. Several species of parasitic nematodes have been shown to express genes encoding orthologues of the mammalian MIF that appear to play a key role in immune evasion by modifying the activity of host cells. In addition, MIF accumulates in nematode somatic cells where its role has not yet been defined. In order to identify the role that MIF plays in the cell biology of nematodes, we have characterized the members of the mif gene family in the free-living species Caenorhabditis elegans. Unlike the single mif gene found in humans and mice, C. elegans expresses four distinct mif genes: Ce-mif-1, Ce-mif-2, Ce-mif-3 and Ce-mif-4. The Ce-MIF proteins are between 15-30% identical to each other, 34-38% identical to the MIFs from the parasitic nematode Brugia malayi, and 22-35% identical to mammalian MIFs. The transcription of Ce-mif-2 and Ce-mif-3, but not Ce-mif-1, was upregulated >100-fold compared to L2 levels when the worms entered the dauer stage. The transcription levels of Ce-mif-2 and Ce-mif-3 fell to near baseline a few hours after exit from dauer. Ce-MIF/GFP transgenic animals and immunostaining were used to demonstrate that the main sites of MIF production are in the hypodermis, body wall muscles and in the nuclei of developing embryos. The results suggest a role for C. elegans MIF in cellular maintenance during periods of adverse conditions that lead to developmental arrest.

OBJECTIVE

Many adipokines, inflammatory cytokines, and other proteins produced by adipose tissue have been shown to be involved in the development of obesity-related insulin resistance. Nevertheless, new factors that play an important role in these processes are still emerging. Therefore, we screened the level of 120 different proteins in biopsies of subcutaneous adipose tissue (ScAT) of lean and obese subjects.

METHODS

All studied volunteers (12 obese with BMI >30 and 6 lean with BMI <25 kg/m(2)) were young, clinically healthy, and drug-naive males with normal glucose tolerance. The ScAT was obtained by a needle biopsy from the umbilical region. Protein levels were assessed in adipose tissue lysates using protein arrays; mRNA levels were determined with the aid of real-time reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

The obese subjects had higher fasting plasma glucose (although within the normal range) and insulin levels, increased high sensitivity C-reactive protein (hsCRP) in circulation, and decreased in vivo insulin action. Using the protein array technique, it was shown that of 120 proteins measured, 27 showed higher levels (leptin, HGF, EGF-R, FGF-6, IGF-1sR, Fas/Apo-1, ENA-78, PARC, lymphotactin, HCC-4, IL-10, IL-1a, IL-1R1, IL-1R4, IL-12p70, angiopoietin-2, Axl, Dtk, MIF, MIP-1a, -1b, -3b, MSP-a, osteoprotegerin, TECK, TIMP-1, -2) and only one (RANTES) showed a lower level in ScAT of obese subjects when compared with the lean controls (p < 0.05). The real-time RT-PCR confirmed the results of protein arrays for leptin, MIF, MIP-1a, TIMP-2, adiponectin, IL-6, and TNF-alpha but not for RANTES.

CONCLUSIONS

To our knowledge, this is the first protein array data on a very early dysregulation of ScAT protein levels in insulin-resistant obese, but apparently healthy, subjects with normal glucose tolerance.

BACKGROUND

Diabetes is associated with an increased risk of cardiovascular disease and atherosclerosis. Increasing evidence shows that CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The purpose of this study was to assess whether CD40 system expressions were disrupted in patients with diabetes.

METHODS

Sixteen normal controls and 72 patients including 20 with type 2 diabetes mellitus (DM), 15 with type 1 DM, 20 with coronary heart disease (CHD) and 17 CHD with coexisting DM were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and serum-soluble CD40L level was determined by a commercially available ELISA. Serum of AGE was detected by fluorescence spectroscopy.

RESULTS

Type 1 DM, type 2 DM, CHD and CHD Patients with coexisting diabetes showed a significant increase of CD40 (81.8 +/- 11.7, 70.7 +/- 11.6, 68.5 +/- 10.2, 79.9 +/- 11.9 MIF, respectively) and CD40L (18.4 +/- 5.1, 13.9 +/- 4.1, 13.5 +/- 3.7, 16.7 +/- 4.7 MIF, respectively) coexpression on platelets as well as sCD40L (15.6 +/- 3.5, 14.1 +/- 3.3, 12.2 +/- 3.5, 13.5 +/- 3.6 ng/ml, respectively) compared with controls (p < 0.01). A positive correlation was found between serum AGE levels in patients with DM and CD40-CD40L system. We also observed a significant correlation between hemoglobinA1c (HbA1c) concentration and CD40L on platelets (r = 0.71, p < 0.001) as well as sCD40L (r = 0.69, p < 0.001), but not for CD40 on platelets.

CONCLUSIONS

Patients with diabetes show increased coexpression of CD40 system, especially CD40L, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis.

OBJECTIVE

Substance P (SP), a neuropeptide mediator of neurogenic inflammation, induces vasodilatation, plasma extravasation and hypersensitivity in the bladder. SP induced inflammation is enhanced and maintained by the release of additional mediators. The rat urothelium contains pre-formed macrophage migration inhibitory factor (MIF), a known proinflammatory cytokine, suggesting that it may mediate bladder neurogenic inflammation. We documented the time course of SP effects on rat bladder inflammation.

METHODS

Subcutaneous SP administration induced neurogenic inflammation. The bladder, urine and serum were removed 15, 30, 60 and 120 minutes following treatment, and changes in MIF, nerve growth factor (NGF), c-fos and cox-2 were determined.

RESULTS

SP induced significant MIF and NGF release from the bladder following 30 minutes of exposure. cox-2 protein was detected at significant levels following 60 minutes of SP exposure. Basal c-fos protein could be detected in control bladders with significant increases following 60 minutes of SP exposure. Histological examination of bladder tissue showed increased edema in SP treated bladders.

CONCLUSIONS

SP stimulated early release of urothelial MIF as well as increased MIF gene expression in this model of neurogenic inflammation. SP also increased expression of the proinflammatory mediator NGF. In addition, increases in cox-2 enzyme and c-fos transcription factor were noted. The early release of MIF suggests that it is an immediate proinflammatory regulator in the bladder and it establishes MIF as candidate proinflammatory mediator of SP induced neurogenic inflammation. These data continue to support our hypothesis that MIF is a new target for intervention in bladder inflammation.

Bronchopulmonary dysplasia (BPD) is a common adverse outcome of prematurity, causing severe morbidity and mortality. The cytokine macrophage migration inhibitory factor (MIF) has been recently shown to favor murine fetal lung development. In this prospective study, we evaluate the expression of MIF in the lung and in the serum of preterm infants (n = 50) and investigate whether the -173 G/C MIF promoter polymorphism is associated with the risk of BPD (n = 103). MIF was highly expressed in lung tissue from preterm infants. Serum MIF levels were measured by ELISA at d 1 after birth. MIF levels were increased [median (interquartile range), 71.01 (44.9-162.3) ng/mL], particularly in those infants with RDS [110.4 (59.4-239.2) ng/mL] compared with healthy adults [2.4 (1.2-5.0) ng/mL], (p < 0.001). The MIF -173*C allele, which predisposes to higher MIF production, was associated with a lower incidence of BPD (OR, 0.2; 95% CI, 0.04-0.93), independently from mechanical ventilation and oxygen exposure (p = 0.03). In conclusion, these data show that MIF expression is increased in lung and serum of preterm infants and suggest that the high producing MIF -173*C allele may be a protective factor for BPD.

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.

The objective of this study was to determine if macrophage migration inhibitory factor (MIF) is upregulated in the bladder during persistent cystitis. MIF is a pro-inflammatory cytokine found pre-formed in the urothelium. Previous findings showed that acute bladder inflammation increased MIF release into the bladder lumen while upregulating MIF and CD74 (MIF receptor) in the bladder. Because the effects of persistent cystitis on MIF and CD74 are not known, MIF and CD74 changes in the bladder were examined after short-term (1-day) or persistent (8-day) cyclophosphamide (CYP)-induced bladder inflammation. Anesthetized male Sprague-Dawley rats received either a single CYP treatment (150 mg/kg, ip; saline, control) and examined 1 day after treatment (short-term), or repeated CYP doses (20-75 mg/ kg, ip; saline, control; every third day for 8 days) and examined after 8 days of treatment (persistent). MIF protein levels in urine and bladder were determined. In addition, Mif, CD74, and cox-2 expression in the bladder was determined. Histology verified cystitis and MIF and CD74 immunoreactivity in the bladder. Repeated CYP doses were decreased to avoid toxicity. Short-term or repeated low CYP doses (40 mg/kg; 8 days) increased urinary MIF and decreased bladder MIF amounts while upregulating bladder Mif and CD74 mRNA expression. Persistent CYP-induced bladder inflammation (even at 40 mg/kg; 8-day treatment) also upregulated other inflammatory cytokines (CCL5, IL-11, iNOS) in the bladder. Short-term and persistent (low dose) CYP cystitis are associated with markedly increased MIF release into the urine and upregulation of Mif and CD74 in bladder. This supports the hypothesis that MIF and CD74 play a significant role in both acute and persistent stages of bladder inflammation.

Myxoid soft-tissue sarcomas represent a heterogeneous group of mesenchymal tumors characterized by a predominantly myxoid matrix, including myxoid liposarcoma (MLS), low-grade fibromyxoid sarcoma (LGFMS), extraskeletal myxoid chondrosarcoma (EMC), myxofibrosarcoma, myxoinflammatory fibroblastic sarcoma (MIFS), and myxoid dermatofibrosarcoma protuberans (DFSP). Cytogenetic and molecular genetic analyses have shown that many of these sarcomas are characterized by recurrent chromosomal translocations resulting in highly specific fusion genes (e.g., FUS-DDIT3 in MLS, FUS-CREB3L2 in LGFMS, EWSR1-NR4A3 in EMC, and COL1A1-PDGFB in myxoid DFSP). Moreover, recent molecular analysis has demonstrated a translocation t(1; 10)(p22; q24) resulting in transcriptional upregulation of FGF8 and NPM3 in MIFS. Most recently, the presence of TGFBR3 and MGEA5 rearrangements has been identified in a subset of MIFS. These genetic alterations can be utilized as an adjunct in diagnostically challenging cases. In contrast, most myxofibrosarcomas have complex karyotypes lacking specific genetic alterations. This paper focuses on the cytogenetic and molecular genetic findings of myxoid soft-tissue sarcomas as well as their clinicopathological characteristics.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine implicated in the pathogenesis of a number of human diseases including inflammatory neurological diseases. Its role in the pathogenesis of cerebral malaria is unknown. Cerebral malaria is a life-threatening complication of falciparum malaria with approximately 20%-30% of patients dying despite appropriate anti-malarial treatment. The reason for this cerebral malaria mortality is still unknown although host proinflammatory factors have been shown to be evidently important. The current study investigated the role of circulating MIF in the pathogenesis and outcomes of cerebral malaria.

RESULTS

Three categories of subjects contributed to this study: healthy controls subjects, mild malaria patients, and cerebral malaria patients. The cerebral malaria patients were further grouped into cerebral malaria survivors and cerebral malaria non-survivors. MIF levels in the peripheral blood plasma, obtained at the time of enrollment, were measured using standard ELISA methods. In logistic regression on cerebral malaria patients, log MIF levels were found to be significantly associated with fatal outcome (odds ratio 4.0; 95%CI 1.6, 9.8; p = 0.003). In multinomial logistic regression log MIF levels were found to be significantly associated with patient category (p = 0.004).

CONCLUSIONS

This study suggests that elevated levels of MIF in the peripheral blood of cerebral malaria patients may be associated with fatal outcomes.

Proper repair of DNA damage is critical for protecting genomic stability, cellular viability and suppression of tumorigenesis. Both p53-dependent and p53-independent pathways have evolved to coordinate the cellular response following DNA damage. In this review, we highlight the importance of the ubiquitously expressed protein macrophage migration inhibitory factor (MIF) for an appropriate response to DNA damage. We discuss the mechanisms by which MIF affects the activity of the ubiquitin-proteasome system, and how this impacts on the integrity of the genome and on cancer.

OBJECTIVE

To assess the effects of mastic administration on cytokine production of circulating mononuclear cells of patients with active Crohn's disease (CD).

METHODS

The study was conducted in patients with established mildly to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Recruited to a 4 wk treatment with mastic caps (6 caps/d, 0.37 g/cap) were 10 patients and 8 controls, all of who successfully completed the protocol. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), macrophage migration inhibitory factor (MIF) and intracellular antioxidant glutathione (GSH) were evaluated in peripheral blood mononuclear cells (PBMC) before and after treatment.

RESULTS

Treating CD patients with mastic resulted in the reduction of TNF-alpha secretion (2.1 +/- 0.9 ng/mL vs 0.5 +/- 0.4 ng/mL, P = 0.028). MIF release was significantly increased (1.2 +/- 0.4 ng/mL vs 2.5 +/- 0.7 ng/mL, P = 0.026) meaning that random migration and chemotaxis of monocytes/macrophages was inhibited. No significant changes were observed in IL-6, MCP-1 and GSH concentrations.

CONCLUSIONS

This study shows that mastic acts as an immunomodulator on PBMC, acting as a TNF-alpha inhibitor and a MIF stimulator. Although further double-blind, placebo-controlled studies in a large number of patients is required to clarify the role of this natural product, this finding provides strong evidence that mastic might be an important regulator of immunity in CD.

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.

OBJECTIVE

What is the role of microRNA-451 (miR-451) in human endometriotic tissue?

CONCLUSIONS

miR451 expression was elevated in endometriotic lesion tissue. MiR451 modulated the expression of macrophage migration inhibitory factor and limited cell survival.

BACKGROUND

microRNAs are post-transcriptional regulators of gene expression which have been reported to be mis-expressed in endometriotic tissue. The exact pattern of expression and role of miR451 in endometriosis is currently unknown.

METHODS

Thirty women with endometriosis are included in the study.

METHODS

Matched eutopic (N = 30) and endometriotic lesion tissue (N = 43) were collected. miR-451, macrophage migration inhibitory factor (MIF), cyclin E1 (CCNE) and phosphatase and tensin homolog (PTEN) mRNA expression were examined by quantitative real-time (qRT)-PCR while MIF protein expression was evaluated by western blot analysis. miR-451 regulation of MIF in vitro translation was confirmed by 3'untranslated region (UTR) reporter assays and western blot analysis. The effect of miR-451 on cell survival was assessed using a human endometrial epithelial cell line (HES).

RESULTS

Compared with eutopic endometrium, both MIF mRNA and protein were significantly (P < 0.05) decreased in endometriotic lesions and this was associated with a significant (P < 0.05) increase in miR-451 expression. Transfection of HES cells with luciferase reporter constructs for MIF revealed that miR-451 specifically bound to the 3'UTR to regulate expression. Further, forced expression of miR-451 induced a significant (P < 0.05) down-regulation of both MIF mRNA and protein in HES cells which was associated with a significant (P < 0.05) reduction in cell survival. Inhibition of MIF using a specific antagonist verified that reduction of MIF contributes to HES cell survival.

CONCLUSIONS

miR-451 and MIF expression were only examined in tissue from women with endometriosis.

CONCLUSIONS

Our data support the hypothesis that miR-451 is elevated in endometriotic tissue and, through regulating MIF expression, may function to limit endometriotic lesion cell survival.

BACKGROUND

This study was funded by the National Institutes of Health/NICHD by grant NIH HD069043 to W.B.N. The authors have no competing interests.

The numerous endogenous opioid peptides (beta-endorphin, enkephalins, dynorphins ... ) and the exogenous opioids (such as morphine) exert their effects through the activation of receptors belonging to four main types, mu, delta, kappa and epsilon. Opioidergic neurones and opioid receptors are largely distributed centrally and peripherally. It is thus not surprising that opioids have numerous pharmacological effects and that endogenous opioids are thought to be involved in the physiological control of various functions, among which nociception is particularly emphasized. Some opioid targets may be components of homeostatic systems tending to reduce the effects of opioids. "Anti-opioid" properties have been attributed to various peptides, especially cholecystokinin (CCK), neuropeptide FF (NPFF) and melanocyte inhibiting factor (MIF)-related peptides. In addition, a particular place should be attributed, paradoxically, to opioid peptides themselves among the anti-opioid peptides. These peptides can oppose some of the acute effects of opioids, and a hyperactivation of anti-opioid peptidergic neurones due to the chronic administration of opioids may be involved in the development of opioid tolerance and/or dependence. In fact, CCK, NPFF and the MIF family of peptides have complex properties and can act as opioid-like as well as anti-opioid peptides. Thus, "opioid modulating peptides" would be a better term to designate these peptides, which probably participate, together with the opioid systems, in multiple feed-back loops for the maintenance of homeostasis. "Opioid modulating peptides" have generally been shown to act through the activation of their own receptors. For example, CCK appears to exert its anti-opioid actions mainly through the activation of CCK-B receptors, whereas its opioid-like effects seem to result from the stimulation of CCK-A receptors. However, the partial agonistic properties at opioid receptors of some MIF-related peptides very likely contribute to their ability to modulate the effects of opioids. CCK- and NPFF-related drugs have potential therapeutic interest as adjuncts to opioids for alleviating pain and/or for the treatment of opioid abuse.

Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-β1 (TGF-β1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-β1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Delta4MIF, Delta5MIF, Delta6MIF, Delta7MIF, Delta8MIF, and Delta10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central beta sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and Delta2MIF exhibiting 40% activity, and Delta4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.

BACKGROUND

Although familial clustering of functional dyspepsia (FD) has been reported, the role of genetics in the susceptibility to FD is still not well established. Several reports indicate the associations between FD and gene polymorphisms, however the data are inconsistent. This review summarized the evidence of genetics in FD based on genetic epidemiology.

RESULTS

Genetic association studies with FD symptom phenotype have limited for several candidate genes investigated. There have been no genome wide association studies in FD. G-protein beta3 (GNB3) subunit C825T was first reported as a candidate gene for FD susceptibility. However, the data are inconsistent in countries. Significant link between homozygous 825C allele of GNB3 protein and dyspepsia was reported from Germany and the USA. On the other hand, the association between T allele of GNB3 C825T polymorphism and dyspepsia was reported from Japan and Netherlands. Association of serotonin transporter promoter (SERT-P) gene polymorphism and FD was reported negatively from a USA community and Netherlands. However we found that SERT SL genotype was significantly associated with PDS. Involvement of IL-17F, migration inhibitory factor (MIF), catechol-o-methyltransferase (COMT) gene val158met, 779 TC of CCK-1 intron 1, cyclooxygenase-1 (COX-1), transient receptor potential cation channel, subfamily V, member 1 (TRPV1) 315C and regulated upon activation normal T cell expressed and secreted (RANTES) polymorphisms was reported in Japanese studies.

CONCLUSIONS

Genetic factors are associated with the development of dyspeptic symptoms. Further studies are needed to confirm these data and to determine how genetic factors influence the clinical manifestation of FD patients.

Myocardial infarction (MI) provokes regional inflammation which facilitates the healing, whereas excessive inflammation leads to adverse cardiac remodelling. Our aim was to determine the role of macrophage migration inhibitory factor (MIF) in inflammation and cardiac remodelling following MI. Wild type (WT) or global MIF deficient (MIFKO) mice were subjected to coronary artery occlusion. Compared to WT mice, MIFKO mice had a significantly lower incidence of post-MI cardiac rupture (27% vs. 53%) and amelioration of cardiac remodelling. These were associated with suppressed myocardial leukocyte infiltration, inflammatory mediators' expression, and reduced activity of MMP-2, MMP-9, p38 and JNK MAPK. Infarct myocardium-derived or exogenous MIF mediated macrophage chemotaxis in vitro that was suppressed by inhibition of p38 MAPK or NF-κB. To further dissect the role of MIF derived from different cellular sources in post-MI cardiac remodelling, we generated chimeric mice with MIF deficiency either in bone marrow derived-cells (WT(KO)) or in somatic-cells (KO(WT)). Compared to WT and KO(WT) mice, WT(KO) mice had reduced rupture risk and ameliorated cardiac remodelling, associated with attenuated regional leukocyte infiltration and expression of inflammatory mediators. In contrast, KO(WT) mice had delayed healing and enhanced expression of M1 macrophage markers, but diminished expression of M2 markers during the early healing phase. In conclusion, global MIF deletion protects the heart from post-infarct cardiac rupture and remodelling through suppression of leukocyte infiltration and inflammation. Leukocyte-derived MIF promotes inflammatory responses after MI, whereas cardiac-derived MIF affects early but not ultimate healing process.

OBJECTIVE

Statins reduce atherosclerosis, but it is controversial whether they suppress abdominal aortic aneurysm (AAA) expansion. We hypothesized that statins (rosuvastatin and atorvastatin) would attenuate angiotensin II (AngII)-induced atherosclerosis and AAA.

RESULTS

Sixty apoE-/- male mice fed a normal diet were administered with either rosuvastatin (10mg/kg/day) or atorvastatin (20mg/kg/day) through drinking water for 1 week prior to initiating 28-day AngII infusion (1000 ng/kg/min). Statins administration led to therapeutic serum concentrations of drugs. Administration of either rosuvastatin or atorvastatin exerted no significant effect on AngII-induced expansion of suprarenal diameter or area. However, atorvastatin significantly reduced AngII-augmented atherosclerotic lesion areas in intimas of both aortic arches and cross-sections of aortic roots (P<0.001). Atherosclerosis was attenuated independent of reductions in serum total cholesterol concentrations. Although serum MCP-1 and MIF concentrations were not changed by either statins, atorvastatin administration increased PPAR-α and -γ mRNA abundances and decreased NF-κB p50, p65, MCP-1 and TNF-α mRNA abundances in atherosclerotic lesions.

CONCLUSIONS

This study demonstrated both statins failed to suppress AngII-induced AAA. In contrast, atorvastatin reduced AngII-induced atherosclerosis associated with no change in serum inflammatory markers but a shift to upregulation of anti-inflammatory status in lesions.

The macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. Two functional polymorphisms have been identified in the promoter region of the MIF gene. We attempted to clarify the associations of these polymorphisms with the development of gastric cancer. The study was performed in 229 patients with gastric cancer and 428 subjects with no evidence of gastric malignancies on the upper gastro-duodenal endoscopy. The severity of histological chronic gastritis was classified according to the updated Sydney system. Overall, the 5-CATT carriers had a reduced risk of developing gastric cancer (OR, 0.67; 96% CI, 0.48-0.93; p=0.015), especially the diffuse type cancer. In subjects >60 years, the adjusted risk for gastric cancer among individuals who were -173C carriers was 1.71 (range, 1.03-2.84; p=0.038) compared to the G/G homozygous genotype. The number of 7-CATT alleles was also positively correlated with the development of intestinal type gastric cancer (adjusted OR, 1.70; 95% CI, 1.02-2.58; p=0.043). In subjects <60 years, the 7/7-CATT homozygous genotype was linked with a risk for the progression of atrophic gastritis (adjusted OR, 8.74; 95% CI, 1.31-58.6; p=0.026). In addition, the number of 7-CATT alleles was significantly correlated with the activity and inflammation scores (p=0.010 and 0.030, respectively). Our results suggested that functional promoter polymorphisms of the MIF gene are associated with the progression of gastric mucosal inflammation and the development of mucosal atrophy at an early stage in life and these genotypes may increase the risk for the subsequent development of gastric cancer, especially the intestinal type, in older subjects.

Macrophage migration inhibitory factor (MIF) is an upstream regulator of immune and inflammatory responses; however, its role in Helicobacter pylori (HP)-associated gastritis remains unknown. We infected MIF knockout (KO) and wild-type mice with SS1 HP and found that 2 weeks after infection, MIF and its receptor CD74 were markedly up-regulated in wild-type mice. This up-regulation preceded the up-regulation of both tumor necrosis factor-alpha and intercellular adhesion molecule-1, as well as the development of moderate gastritis at 8 weeks, as determined by a significant infiltration of neutrophils, T cells, and macrophages. In contrast, KO mice were protected against HP-induced gastritis by preventing the up-regulation of CD74 and Th1-mediated immune injury, including a reduction in the Th1 transcriptional factor T-bet and the expression of interferon-gamma. Additionally, inhibition of skin delayed type hypersensitivity reactions to HP antigens in KO mice also suggested a critical role for MIF in cell-mediated injury. A regulatory role for MIF in Th1-immune responses was further demonstrated by the finding that antigen-primed CD4(+) T cells lacking MIF failed to differentiate into the Th1 phenotype; these cells were instead promoted to Th2 differentiation after challenge with HP antigen in vitro. Results from this study indicated that inhibition of HP-induced innate immune responses and Th1-mediated immune injury may be the key mechanisms by which KO mice failed to develop gastritis after HP infection.

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD.