BACKGROUND

Several studies have shown that serum IL-10, IFNγ and MIF are elevated in patients in severe dengue (SD) and could be used as potential biomarkers. We proceeded to determine if these cytokines could be used as biomarkers in a large cohort of adult dengue patients with varying severity of dengue infection.

METHODS

Serum IL-10 levels were determined in 259 of whom 40 had severe dengue infection. Serum IFNγ and IFNα levels were done in 78 and MIF levels were done in 65 patients with acute dengue infection. Clinical features and laboratory investigations were undertaken during the febrile and critical phase.

RESULTS

We found that serum IL-10 levels were significantly higher (p = 0.001) in patients with SD, when compared to those with non SD. Serum IL-10 levels significantly and inversely correlated with white cell counts (R = -0.23, p = 0.0002) and lymphocyte counts (R = -0.29, p < 0.0001) but significantly and positively correlated with aspartate tranaminase levels (R = 0.16, p = 0.01) and alanine transaminase levels (R = 0.22, p = 0.007). However, IL-10 levels did not have a good predictive value in discriminating those who were likely to develop SD (AUC = 0.66). Serum IFNγ levels were also significantly higher (p = 0.04) in patients with SD when compared to non SD. There was no difference (p = 0.34) in serum IFNα levels and serum MIF levels (p = 0.15) in patients with SD and non SD.

CONCLUSIONS

Although serum IL-10 was significantly elevated in patients with SD it had a poor discriminatory value in identifying those with SD and non SD and therefore, is unsuitable to be used as a robust biomarker in this cohort.

Inflammatory processes may substantially contribute to the cerebral pathology in Alzheimer's disease (AD) and accelerate the disease progression. The macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine which promotes the production of several inflammatory mediators such as TNF-alpha, IL-6 and IFN-gamma, and plays a central regulatory role in the pathogenesis of several inflammatory and autoimmune diseases. There is now first evidence that MIF may be involved in the neuroinflammation in AD. To determine whether MIF production is up-regulated early in the course of AD, we compared the levels of MIF assessed by ELISA in the cerebrospinal fluid (CSF) of 31 patients with AD, 28 patients with amnestic mild cognitive impairment (MCI), and 19 subjects without cognitive deficits. Additionally, we measured the CSF concentrations of the inflammatory mediators TNF-alpha, IL-6 and IFN-gamma, which are thought to be both up-regulated by MIF and involved in the pathophysiology of AD. CSF MIF concentrations were significantly increased in AD (p=0.003) and MCI patients (p<0.001) compared to controls. The levels of TNF-alpha, IL-6 and IFN-gamma did not differ significantly between the groups. There was a correlation only between the concentrations of MIF and of TNF-alpha in the AD group (r=0.407; p=0.023). These results demonstrate increased MIF production in AD and MCI suggesting that MIF may be involved in the occurring neuroinflammatory process at a clinical pre-dementia disease stage.

Pharmacophores are chemical scaffolds upon which changes in chemical moieties (R-groups) at specific sites are made to identify a combination of R-groups that increases the therapeutic potency of a small molecule inhibitor while minimizing adverse effects. We developed a pharmacophore based on a carbonyloxime (OXIM) scaffold for macrophage migration inhibitory factor (MIF), a protein involved in the pathology of sepsis, to validate that inhibition of a catalytic site could produce therapeutic benefits. We studied the crystal structures of MIF.OXIM-based inhibitors and found two opposite orientations for binding to the active site that were dependent on the chemical structures of an R-group. One orientation was completely unexpected based on previous studies with hydroxyphenylpyruvate and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1). We further confirmed that the unexpected binding mode targets MIF in cellular studies by showing that one compound, OXIM-11, abolished the counter-regulatory activity of MIF on anti-inflammatory glucocorticoid action. OXIM-11 treatment of mice, initiated 24 h after the onset of cecal ligation and puncture-induced sepsis, significantly improved survival when compared with vehicle-treated controls, confirming that inhibition of the MIF catalytic site could produce therapeutic effects. The crystal structures of the MIF inhibitor complexes provide insight for further structure-based drug design efforts.

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.

Ultrasound (US) contrast agents have gained wide interest in gene therapy as many researchers reported increased membrane permeability and transfection efficiency by sonoporation in the presence of US contrast agents. We recently demonstrated an increase in cell membrane permeability for Ca2+ in rat cardiomyoblast (H9c2) cells insonified in the presence of microbubbles. In the present study, we specifically investigated whether US-exposed microbubbles have an effect on the cell membrane potential and whether Ca2+-dependent potassium (BK(Ca)) channels are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts with a frequency of 1 MHz with a peak-to-peak pressure of 0.1 or 0.5 MPa. Pulse repetition frequency was set to 20 Hz, with a duty cycle of 0.2%. Cells were insonified during 30 s in the presence of Sonovue(trade mark) microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-aminonaphtylethenylpyridinium (di-4-ANEPPS). The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK(Ca) channels. Surprisingly, despite the previously reported Ca(2+) influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean intensity of fluorescence (MIF) to 118.6 +/- 2.5% (p < 0.001, n = 267) at 0.1 MPa and 125.7 +/- 5.9% (p < 0.001, n = 161) at 0.5 MPa at t = 74 s, respectively, compared with "no US" (100.3 +/- 3.4%, n = 52). This hyperpolarization was caused by the activation of BK(Ca) channels, as iberiotoxin completely prevented hyperpolarization. (MIF(t74) = 100.6 +/- 1.4%; p < 0.001, n = 267) and 0.5 MPa (MIF(t74) = 88.8 +/- 2.0%; p< 0.001, n = 193), compared with 0.1 and 0.5 MPa microbubbles without iberiotoxin. In conclusion, US-exposed microbubbles elicit a Ca2+ influx, which leads to activation of BK(Ca) channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. (E-mail: ljm.juffermans@vumc.nl).

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in many inflammatory reactions and disorders, and it has become evident that it also affects glucose homeostasis. The protein is produced by pancreatic beta cells and can promote the release of insulin. It also modulates glucose uptake, glycolysis and insulin resistance in insulin target cells such as the adipocyte, myocyte and cardiomyocyte. Possessing both immunological and endocrinological properties, MIF has been associated with the development of type 1 and type 2 diabetes, and it may be important in the setting of islet transplantation. The present review summarises our current knowledge, based on clinical and research data, on the impact of MIF on both physiological and pathological aspects of glucose metabolism.

The presence of alpha-MSH receptors on human melanoma has so far been suggested in the literature but not proved. We describe a reproducible and specific binding assay of alpha-MSH on human melanoma cells, using a high-specific-activity 125I-labelled hormone (1.5 to 2 mCi/micrograms) with consistent receptor binding (usually exceeding 2 pg/10(6) cells) and stable for 3 weeks. Asynchronized cells in suspension were incubated for 15 min at 37 degrees C with the tracer and various concentrations of unlabelled hormones. Synthetic alpha-MSH was compared to beta-MSH, ACTH1-24, ACTH4-10, beta-LPH, CLIP, CRF, MIF I, A8VP and beta-endorphin. Out of a panel of 8 human melanoma cell lines, 3 showed specific and reproducible alpha-MSH binding curves. No significant binding to human fibroblast and human carcinoma cells was seen. alpha-MSH, beta-MSH and, to a lesser extent ACTH4-10 (a part of the alpha-MSH sequence) were the only peptides able to displace labelled alpha-MSH from its binding sites, indicating the high specificity of the MSH receptor. Affinity constants (Ka) ranged from 10(8) to 10(9) l/mole and the estimated receptor number was 1,000 to 2,000 per cell. We conclude that some human melanoma cell lines expressed specific MSH receptors with stable affinity but which are low in number.

OBJECTIVE

Here, we aimed to clarify the role of CXC chemokine receptor (CXCR) 2 in macrophage migration-inhibitory factor (MIF)-mediated effects after myocardial ischemia and reperfusion. As a pleiotropic chemokine-like cytokine, MIF has been identified to activate multiple receptors, including CD74 and CXCR2. In models of myocardial infarction, MIF exerts both proinflammatory effects and protective effects in cardiomyocytes. Similarly, CXCR2 displays opposing effects in resident versus circulating cells.

RESULTS

Using bone marrow transplantation, we generated chimeric mice with Cxcr2(-/-) bone marrow-derived inflammatory cells and wild-type (wt) resident cells (wt/Cxcr2(-/-)), Cxcr2(-/-) cardiomyocytes and wt bone marrow-derived cells (Cxcr2(-/-)/wt), and wt controls reconstituted with wt bone marrow (wt/wt). All groups were treated with anti-MIF or isotype control antibody before they underwent myocardial ischemia and reperfusion. Blocking MIF increased infarction size and impaired cardiac function in wt/wt and wt/CXCR2(-/-) mice but ameliorated functional parameters in Cxcr2(-/-)/wt mice, as analyzed by echocardiography and Langendorff perfusion. Neutrophil infiltration and angiogenesis were unaltered by MIF blockade or Cxcr2 deficiency. Monocyte infiltration was blunted in wt/Cxcr2(-/-) mice and reduced by MIF blockade in wt/wt and Cxcr2(-/-)/wt mice. Furthermore, MIF blockade attenuated collagen content in all groups in a CXCR2-independent manner.

CONCLUSIONS

The compartmentalized and opposing effects of MIF after myocardial ischemia and reperfusion are largely mediated by CXCR2. Although MIF confers protective effects by improving myocardial healing and function through CXCR2 in resident cells, thereby complementing paracrine effects through CD74/AMP-activated protein kinase, it exerts detrimental effects on CXCR2-bearing inflammatory cells by increasing monocyte infiltration and impairing heart function. These dichotomous findings should be considered when developing novel therapeutic strategies to treat myocardial infarction.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

RESULTS

Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3(rd) day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

CONCLUSIONS

These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine released from T-cells and macrophages. Although a detailed understanding of the biological functions of MIF has not yet been clarified, it is known that MIF catalyzes the tautomerization of a nonphysiological molecule, D-dopachrome. Using a structure-based computer-assisted search of two databases of commercially available compounds, we have found 14 novel tautomerase inhibitors of MIF whose K(i) values are in the range of 0.038-7.4 microM. We also have determined the crystal structure of MIF complexed with the hit compound 1. It showed that the hit compound is located in the active site of MIF containing the N-terminal proline which plays an important role in the tautomerase reaction and forms several hydrogen bonds and undergoes hydrophobic interactions. A crystallographic study also revealed that there is a hydrophobic surface which consists of Pro-33, Tyr-36, Trp-108, and Phe-113 at the rim of the active site of MIF, and molecular modeling studies indicated that several more potent hit compounds have the aromatic rings which can interact with this hydrophobic surface. To our knowledge, our compounds are the most potent tautomerase inhibitors of MIF. One of these small, drug-like molecules has been cocrystallized with MIF and binds to the active site for tautomerase activity. Molecular modeling also suggests that the other hit compounds can bind in a similar fashion.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pathogenic role in experimental crescentic glomerulonephritis (GN). Renal expression of MIF is also upregulated in human GN and correlates with leukocytic infiltration, histologic damage, and renal dysfunction. The study presented here examined whether MIF can be measured in urine and if so, whether the urine MIF concentration reflects the degree of renal injury. Urine and serum MIF was measured by enzyme-linked immunosorbent assay in 10 normal healthy volunteers and in a cohort of 63 patients with GN (2 thin basement membrane disease [TBM], 15 membranous GN, 10 focal segmental glomerular sclerosis, 20 IgA glomerularnephritis, 11 crescentic GN, 10 systemic lupus erythematosis World Health Organization class IV). Renal MIF expression was assessed by immunostaining of biopsy tissue. MIF was detected in urine from normal volunteers (mean +/- SD; 191 +/- 132 pg MIF/micromol creatinine). The urine MIF concentration was unchanged in patients with nonproliferative nephropathies (343 +/- 397 pg MIF/micromol Cr) but was increased 3.4-fold in proliferative nephropathies (645 +/- 527 pg MIF/micromol Cr; P < 0.05 versus normal and nonproliferative). Stratified analysis showed the greatest increase in urine MIF in crescentic GN (4.5-fold). In contrast, serum MIF levels were not different between normal patients and any patient group. Immunostaining demonstrated a significant increase in renal MIF expression in proliferative glomerulonephritides that was associated with macrophage and T cell infiltration. There was a significant correlation between the urine MIF concentration and renal MIF expression, but not with serum MIF, indicating a renal origin for the excreted urine MIF. The urine MIF concentration also correlated with the degree of renal dysfunction, histologic damage, and leukocytic infiltration, but not with the amount of proteinuria. In conclusion, this study shows that the urine MIF concentration is significantly increased in proliferative forms of GN and correlates with the degree of renal injury. Urine MIF levels reflect MIF expression within the kidney and may be a useful noninvasive tool for monitoring patients with crescentic GN, particularly in disease exacerbation.

Acute myocardial infarction (AMI) remains one of the leading causes of death in the developed world. There is emerging evidence that the cytokine macrophage migration inhibitory factor (MIF) is a crucial player in AMI. Cardioprotection by MIF is likely to be a multifactorial phenomenon mediated by receptor-mediated signalling processes, intracellular protein-protein interactions, and enzymatic redox regulation. Co-ordinating several pathways in the ischaemic heart, MIF contributes to receptor-mediated regulation of cardioprotective AMP-activated protein kinase signalling, inhibition of pro-apoptotic cascades, and the reduction of oxidative stress in the post-ischaemic heart. Moreover, the cardioprotective properties of MIF are modulated by S-nitros(yl)ation. These effects in the pathophysiology of myocardial ischaemia/reperfusion injury qualify MIF as a promising therapeutic target in the future. We here summarize the findings of experimental and clinical studies and emphasize the therapeutic potential of MIF in AMI.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by renal cyst formation, inflammation, and fibrosis. Macrophages infiltrate cystic kidneys, but the role of these and other inflammatory factors in disease progression are poorly understood. Here, we identified macrophage migration inhibitory factor (MIF) as an important regulator of cyst growth in ADPKD. MIF was upregulated in cyst-lining epithelial cells in polycystin-1-deficient murine kidneys and accumulated in cyst fluid of human ADPKD kidneys. MIF promoted cystic epithelial cell proliferation by activating ERK, mTOR, and Rb/E2F pathways and by increasing glucose uptake and ATP production, which inhibited AMP-activated protein kinase signaling. MIF also regulated cystic renal epithelial cell apoptosis through p53-dependent signaling. In polycystin-1-deficient mice, MIF was required for recruitment and retention of renal macrophages, which promoted cyst expansion, and Mif deletion or pharmacologic inhibition delayed cyst growth in multiple murine ADPKD models. MIF-dependent macrophage recruitment was associated with upregulation of monocyte chemotactic protein 1 (MCP-1) and inflammatory cytokine TNF-α. TNF-α induced MIF expression, and MIF subsequently exacerbated TNF-α expression in renal epithelial cells, suggesting a positive feedback loop between TNF-α and MIF during cyst development. Our study indicates MIF is a central and upstream regulator of ADPKD pathogenesis and provides a rationale for further exploration of MIF as a therapeutic target for ADPKD.

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.

BACKGROUND

Chemerin is a specific chemoattractant for macrophages and dendritic cells (DC). In addition, it can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules and is able to activate fibroblast-like synoviocytes (FLS), suggesting a role in the pathogenesis of rheumatoid arthritis (RA). Chemerin is also an adipocytokine that has been related to the inflammatory state of endothelial cells and as such could be involved in the changes in endothelial cells in RA and perhaps increased cardiovascular morbidity. We investigated whether anti-Tumor Necrosis Factor (TNF) treatment affects chemerin levels.

METHODS

49 patients with active RA (disease activity score evaluated in 28 joints (DAS28) ≥3.2) were started on adalimumab therapy. Blood was drawn from patients while fasting at baseline and 16 weeks after initiation of treatment. Chemerin serum levels were measured by ELISA and related to disease activity, mediators of inflammation and known risk factors for cardiovascular disease.

RESULTS

Adalimumab therapy reduced chemerin serum levels, which was correlated with the reduction in DAS28 (r = 0.37, p = 0.009). In addition, the decrease in chemerin serum levels after anti-TNF treatment was associated with the decrease in serum levels of IL-6 (r = 0.39, p = 0.033) and macrophage migration inhibitory factor (MIF) (r = 0.31, p = 0.049). Baseline chemerin serum levels were not related to traditional risk factors for atherosclerosis, except perhaps for smoking (p = 0.07).

CONCLUSIONS

This exploratory study shows that adalimumab therapy lowers chemerin levels, which is associated with the reduction in disease activity parameters, and inflammatory mediators IL-6 and MIF. This suggests a possible involvement of chemerin in the migration/retention of macrophages in the synovium. TRIAL REGISTRATION NEDERLANDS TRIAL REGISTER: NTR 857.

The novel avian origin influenza A (H7N9) virus has caused severe diseases in humans in eastern China since the spring of 2013. Fatal outcomes of H7N9 infections are often attributed to the severe pneumonia and acute respiratory distress syndrome (ARDS). There is urgent need to discover biomarkers predicting the progression of disease and fatal outcome of potentially lethal flu infections, based on sound statistical analysis. We discovered that 34 of the 48 cytokines and chemokines examined in this study were significantly elevated in the plasma samples from patients infected with H7N9. We report for the first time that the levels of MIF, SCF, MCP-1, HGF, and SCGF-β are highly positively linked to disease severity and the profile of mediators MIF, SCF, MCP-1, HGF, SCGF-β, IP-10, IL-18, and IFN-γ is an independent outcome predictor.

OBJECTIVE

Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined.

METHODS

EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry.

RESULTS

In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction.

CONCLUSIONS

Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

BACKGROUND

Inflammation and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). CD74 is a receptor for the proinflammatory cytokine macrophage migration inhibitory factor (MIF). This ligand/receptor complex initiates survival pathways and cell proliferation, and it triggers the synthesis and secretion of major proinflammatory factors and cell adhesion molecules.

OBJECTIVE

We hypothesized that the MIF/CD74 signaling pathway is overexpressed in idiopathic PAH (iPAH) and contributes to a proinflammatory endothelial cell (EC) phenotype.

METHODS

Primary early passage cultures of human ECs isolated from lung tissues obtained from patients with iPAH and controls were examined for their ability to secrete proinflammatory mediators and bind inflammatory cells with or without modulation of the functional activities of the MIF/CD74 complex. In addition, we tested the efficacies of curative treatments with either the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies on the aberrant proinflammatory EC phenotype in vitro and in vivo and on the progression of monocrotaline-induced pulmonary hypertension.

RESULTS

In human lung tissues, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions are markedly up-regulated in the endothelium of distal iPAH pulmonary arteries. Circulating MIF levels are increased in the serum of patients with PAH compared with control subjects, and T-cell lymphocytes represent a source of this overabundance. In addition, CD74 is highly expressed in the endothelium of muscularized pulmonary arterioles and in cultured pulmonary ECs from iPAH, contributing to an exaggerated recruitment of peripheral blood mononuclear cells to pulmonary iPAH ECs. Finally, we found that curative treatments with the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies partially reversed development of pulmonary hypertension in rats and substantially reduced inflammatory cell infiltration.

CONCLUSIONS

We report here that CD74 and MIF are markedly increased and activated in patients with iPAH, contributing to the abnormal proinflammatory phenotype of pulmonary ECs in iPAH.

SiteHound uses Molecular Interaction Fields (MIFs) produced by EasyMIFs to identify protein structure regions that show a high propensity for interaction with ligands. The type of binding site identified depends on the probe atom used in the MIF calculation. The input to EasyMIFs is a PDB file of a protein structure; the output MIF serves as input to SiteHound, which in turn produces a list of putative binding sites. Extensive testing of SiteHound for the detection of binding sites for drug-like molecules and phosphorylated ligands has been carried out.

BACKGROUND

EasyMIFs and SiteHound executables for Linux, Mac OS X, and MS Windows operating systems are freely available for download from http://sitehound.sanchezlab.org/download.html.

BACKGROUND

Supplementary data are available at Bioinformatics online.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum) levels. Recent studies from another laboratory failed to document these results. This study's aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate.

METHODS

MIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis.

RESULTS

Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87 +/- 3.91 ng/ml; +/- interquartile range; n = 115) compared with patients with no documented diagnosis of prostate cancer (2.19 +/- 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected in culture medium from prostate cancer cell lines (LNCaP and PC-3).

CONCLUSIONS

Increased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer.

The invariant chain (CD74) is well known for its essential role in antigen presentation by mediating assembly and subcellular trafficking of the MHCII complex. Beyond this, CD74 has also been implicated in a number of processes independent of MHCII. These include the regulation of endosomal trafficking, cell migration and cellular signalling as surface receptor of the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). In several forms of cancer, CD74 is up-regulated and associated with enhanced proliferation and metastatic potential. In this review, an overview of the diverse biological functions of the CD74 protein is provided with a particular focus on how these may be regulated. In particular, proteolysis of CD74 will be discussed as a central mechanism to control the actions of this important protein at different levels.

OBJECTIVE

To investigate the relationship of aqueous macrophage migration inhibitory factor (MIF) and monocyte chemotactic protein-1 (MCP-1) levels with the clinical stage of diabetic retinopathy.

METHODS

We assayed MIF and MCP-1 levels in aqueous humour samples obtained from 40 diabetic patients (49 eyes) and 24 non-diabetic patients (31 eyes) using enzyme-linked immunosorbent assay. According to the clinical stage of diabetic retinopathy, the diabetic patients were classified into non-diabetic retinopathy (11 eyes), non-proliferative diabetic retinopathy (14 eyes) and proliferative diabetic retinopathy (24 eyes).

RESULTS

The aqueous levels of MIF (mean +/- sd) were 6.34 +/- 4.53 ng/ml in proliferative diabetic retinopathy, 3.22 +/- 1.71 ng/ml in non proliferative diabetic retinopathy, 1.25 +/- 0.96 ng/ml in non-diabetic retinopathy and 1.07 +/- 0.94 ng/ml in non-diabetic patients. Significant differences were found among these four groups (P < 0.0001). Aqueous MCP-1 levels were 1668.6 +/- 1442.3 pg/ml in proliferative diabetic retinopathy, 1528.6 +/- 1994.6 pg/ml in non-proliferative diabetic retinopathy, 690.2 +/- 402.1 pg/ml in non-diabetic retinopathy and 622.7 +/- 245.3 pg/ml in non-diabetic patients. Significant differences were also found among these four groups (P < 0.0001). After correcting for total aqueous protein, the ratios of MIF and MCP-1 to total protein remained significantly correlated with the clinical stage of diabetic retinopathy (P < 0.0001, P = 0.0004, respectively). The ratios of MIF to total protein significantly correlated with the ratios of MCP-1 to total protein in diabetic patients (r = 0.680, P < 0.0001).

CONCLUSIONS

Aqueous MIF levels significantly correlated with aqueous MCP-1 levels and the clinical stage of diabetic retinopathy. The results suggest that MIF has a co-operative role with MCP-1 in the progression of diabetic retinopathy.

A brief description of current procedures for the Chlamydia microimmunofluorescence (MIF) test is presented. To date, use of MIF serology with Chlamydia pneumoniae (TWAR) antigen has provided the most sensitive and specific method for diagnosis of acute TWAR infection. In primary infections, the TWAR IgM antibody response is longer lasting and IgG antibody is slower to develop compared with Chlamydia trachomatis infection. Unlike other Chlamydia species, only a single serovar for C. pneumoniae has been recognized in the MIF system and cross-reaction with other species is negligible. While IgM antibody response is an important marker for serodiagnosis of acute infection, rheumatoid factor often causes false-positive reactions. Persistent TWAR IgG antibody has been useful for seroepidemiologic studies and an association of TWAR IgG antibody and atherosclerotic diseases has been observed. IgA antibody may not be a useful marker for chronic TWAR infection or for acute infection.