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Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a "near cure") with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue., (Published by Elsevier Inc.)

Type 2 diabetes mellitus (T2D) contributes to sustained inflammation and myopathic changes in the heart although the precise interplay between the two remains largely unknown. This study evaluated the impact of deficiency in CD74, the cognate receptor for the regulatory cytokine macrophage migration inhibitory factor (MIF), in T2D-induced cardiac remodeling and functional responses, and cell death domains involved. WT and CD74 -/- mice were fed a high fat diet (60% calorie from fat) for 8 weeks prior to injection of streptozotocin (STZ, 35 mg/kg, i.p., 3 consecutive days) and were maintained for another 8 weeks. KEGG analysis for differentially expressed genes (DEGs) reported gene ontology term related to ferroptosis in T2D mouse hearts. T2D patients displayed elevated plasma MIF levels. Murine T2D exerted overt global metabolic derangements, cardiac remodeling, contractile dysfunction, apoptosis, pyroptosis, ferroptosis and mitochondrial dysfunction, ablation of CD74 attenuated T2D-induced cardiac remodeling, contractile dysfunction, various forms of cell death and mitochondrial defects without affecting global metabolic defects. CD74 ablation rescued T2D-evoked NLRP3-Caspase1 activation and oxidative stress but not dampened autophagy. In vitro evidence depicted that high glucose/high fat (HGHF) compromised cardiomyocyte function and promoted lipid peroxidation, the effects were ablated by inhibitors of NLRP3, pyroptosis, and ferroptosis but not by the mitochondrial targeted antioxidant mitoQ. Recombinant MIF mimicked HGHF-induced lipid peroxidation, GSH depletion and ferroptosis, the effects of which were reversed by inhibitors of MIF, NLRP3 and pyroptosis. Taken together, these data suggest that CD74 ablation protects against T2D-induced cardiac remodeling and contractile dysfunction through NLRP3/pyroptosis-mediated regulation of ferroptosis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF-1) and its homologue d-dopachrome tautomerase (MIF-2) share the common CD74 receptor and function innately to enhance severity of multiple sclerosis (MS) as well as the experimental autoimmune encephalomyelitis (EAE) model for MS. We previously demonstrated that genetically high-MIF-expressing male subjects with relapsing MS had a significantly greater risk of conversion to progressive MS (PMS) than lower-MIF-expressing males. To expand on this observation, we utilized MIF-1, MIF-2, and MIF-1/2-DUAL-deficient male mice to discern if there would be a greater contribution of these inflammatory factors in EAE mice with severe vs. moderate clinical disease signs. As shown previously, mice deficient in either MIF-1 or MIF-2 each had a ∼25% reduction of moderate EAE compared to WT mice, with significant differences in disease onset and trajectory. However, EAE induction in mice deficient in both MIF-1 and MIF-2 genes did not result in a further reduction in EAE severity. This result suggests that the two MIF homologues were likely affecting the same pathogenic pathways such that each could partially compensate for the other but not in an additive or synergistic manner. However, MIF-1-KO, MIF-2-KO, and MIF-1/2-DUAL-KO mice with severe EAE did not exhibit a significant reduction in cumulative EAE scores compared with WT mice, but the MIF-1-KO and, to a lesser extent, MIF-1/2-DUAL-KO mice did show a significant reduction in daily EAE scores over the last 3 days of observation, and MIF-2-KO mice showed a more modest but still consistent reduction over the same span. Furthermore, deletion of MIF-1 resulted in a massive reduction in the expression of EAE- and Complete Freund's Adjuvant-associated inflammatory factors, suggesting delayed involvement of the MIF/CD74 axis in promoting disease expression. To further explore modulation of MIF-1 and MIF-2 effects on EAE, we treated WT mice with moderate EAE using DRα1-mMOG-35-55, an inhibitor of CD74 that blocks both MIF-1 and MIF-2 action. This treatment reduced ongoing moderate EAE severity in excess of 25%, suggesting efficient blockade of the MIF/CD74 axis in disease-enhancing pathways. Moreover, DRα1-mMOG-35-55 treatment of mice with severe EAE strongly reversed EAE- and CFA-associated expression of inflammatory cytokines and chemokines including Tnf, Ccr7, Ccr6, Ccl8, Cxcr3, and Ccl19 in MIF-deficient mouse genotypes, and also exceeded innate MIF-1 and MIF-2 EAE enhancing effects, especially in MIF-1-KO mice. These results illustrate the therapeutic potential of targeting the disease-enhancing MIF/CD74 pathway in male mice with moderate and severe EAE, with implications for treatment of high-MIF-expressing RRMS human males at risk of conversion to progressive MS as well as those that have already transitioned to PMS., (Published by Elsevier Inc.)

Background: Tocilizumab, an IL-6 receptor antagonist, can be used to treat cytokine release syndrome (CRS), with observed improvements in a coronavirus disease 2019 (COVID-19) case series., Research Question: The goal of this study was to determine if tocilizumab benefits patients hospitalized with COVID-19., Study Design and Methods: This observational study of consecutive COVID-19 patients hospitalized between March 10, 2020, and March 31, 2020, and followed up through April 21, 2020, was conducted by chart review. Patients were treated with tocilizumab using an algorithm that targeted CRS. Survival and mechanical ventilation (MV) outcomes were reported for 14 days and stratified according to disease severity designated at admission (severe, ≥ 3 L supplemental oxygen to maintain oxygen saturation > 93%). For tocilizumab-treated patients, pre/post analyses of clinical response, biomarkers, and safety outcomes were assessed. Post hoc survival analyses were conducted for race/ethnicity., Results: Among the 239 patients, median age was 64 years; 36% and 19% were black and Hispanic, respectively. Hospital census increased exponentially, yet MV census did not. Severe disease was associated with lower survival (78% vs 93%; P < .001), greater proportion requiring MV (44% vs 5%; P < .001), and longer median MV days (5.5 vs 1.0; P = .003). Tocilizumab-treated patients (n = 153 [64%]) comprised 90% of those with severe disease; 44% of patients with nonsevere disease received tocilizumab for evolving CRS. Tocilizumab-treated patients with severe disease had higher admission levels of high-sensitivity C-reactive protein (120 vs 71 mg/L; P < .001) and received tocilizumab sooner (2 vs 3 days; P < .001), but their survival was similar to that of patients with nonsevere disease (83% vs 91%; P = .11). For tocilizumab-treated patients requiring MV, survival was 75% (95% CI, 64-89). Following tocilizumab treatment, few adverse events occurred, and oxygenation and inflammatory biomarkers (eg, high-sensitivity C-reactive protein, IL-6) improved; however, D-dimer and soluble IL-2 receptor (also termed CD25) levels increased significantly. Survival in black and Hispanic patients, after controlling for age, was significantly higher than in white patients (log-rank test, P = .002)., Interpretation: A treatment algorithm that included tocilizumab to target CRS may influence MV and survival outcomes. In tocilizumab-treated patients, oxygenation and inflammatory biomarkers improved, with higher than expected survival. Randomized trials must confirm these findings., (Published by Elsevier Inc.)

Second hand smoke exposure increases the prevalence of chronic diseases partly attributed to inflammatory responses. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is involved in the pathogenesis of multiple diseases although its role in second hand smoke exposure-induced cardiac anomalies remains elusive. This study evaluated the impact of MIF knockout on side-stream smoke exposure-induced cardiac pathology and underlying mechanisms. Adult WT and MIF knockout (MIFKO) mice were placed in a chamber exposed to cigarette smoke for 1 h daily for 60 consecutive days. Echocardiographic, cardiomyocyte function and intracellular Ca 2+ handling were evaluated. Autophagy, mitophagy and apoptosis were examined using western blot. DHE staining was used to evaluate superoxide anion (O 2 - ) generation. Masson trichrome staining was employed to assess interstitial fibrosis. Our data revealed that MIF knockout accentuated side-stream smoke-induced cardiac anomalies in fractional shortening, cardiomyocyte function, intracellular Ca 2+ homeostasis, myocardial ultrastructure and mitochondrial content along with overt apoptosis and O 2 - generation. In addition, unfavorable effects of side-stream smoke were accompanied by excessive formation of autophagolysosome and elevated TFEB, the effect of which was exacerbated by MIF knockout. Recombinant MIF rescued smoke extract-induced myopathic anomalies through promoting AMPK activation, mitophagy and lysosomal function. Taken together, our data suggest that MIF serves as a protective factor against side-stream smoke exposure-induced myopathic changes through facilitating mitophagy and autophagolysosome formation., Competing Interests: Declaration of Competing Interest Yale University hold intellectual property rights for the therapeutic augmentation of MIF signaling in cardiac tissue protection. RB is a co-inventor on this patent and a co-founder of MIFCOR, Inc, which seeks to augment MIF pathways for ischemic tissue injury. RB receives research support from MIFCOR, Inc., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CD74, a non-polymorphic type II transmembrane glycoprotein and MHC class II chaperone, is the cell surface receptor for the inflammatory cytokine macrophage migration inhibitory factor (MIF) and participates in inflammatory signaling regulation. This study examined the potential role of CD74 in binge drinking-induced cardiac contractile dysfunction. WT and CD74 knockout mice were exposed to ethanol (3 g/kg/d, i.p., for 3 days). Echocardiography, cardiomyocyte function, histological staining and autophagy signaling including AMPK, mTOR, and AMPK downstream signals Skp2 and Sirt1 were evaluated. Our results revealed that ethanol challenge overtly compromised echocardiographic, cardiomyocyte contractile, intracellular Ca 2+ and ultrastructural properties along with overt apoptosis, inflammation (elevated MIF, IL-1β and IL-6) and mitochondrial O 2 - production (p < 0.01), the effect of which was reconciled by CD74 ablation (p < 0.01 vs. ethanol group) with the exception of MIF expression. Ethanol challenge upregulated autophagy (p < 0.001), promoted AMPK phosphorylation and Sirt1 levels (p < 0.003) while suppressing mTOR phosphorylation and Skp2 levels (p < 0.02). These effects were reversed by CD74 ablation. In vitro studies demonstrated that short-term ethanol challenge compromised cardiomyocyte contractile function and facilitated GFP-Puncta formation, which were mitigated by CD74 knockout (p < 0.0001). Moreover, the CD74 ablation-offered beneficial effects against ethanol-induced cardiomyocyte dysfunction, and GFP-Puncta formation were nullified by the AMPK activator AICAR, the Skp2 inhibitor C1 or the Sirt1 activator SRT1720 (p < 0.0001). Taken together, our data revealed that CD74 ablation counteracts acute ethanol challenge-induced myocardial dysfunction, inflammation and apoptosis possibly through an AMPK-mTOR-Skp2-mediated regulation of autophagy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Macrophage migration Inhibitory Factor (MIF) is a cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive-feedback-loop with proinflammatory cytokines and could perpetuate the inflammatory process in Systemic Lupus Erythematosus (SLE).The aim of this study was to assess the effect of recombinant-human-MIF (rhMIF) on the expression of Th1, Th2 and Th17 cytokines in Peripheral Blood Mononuclear Cells (PBMC) from Healthy Subjects (HS) and SLE patients. The PBMC were isolated from SLE patients classified according to the 1997 SLE ACR criteria and HS donors; all subjects included were women from an unrelated Mexican-Mestizo population. The PBMC isolated were stimulated with rhMIF, LPS and ISO-1 in different combinations; Th1, Th2 and Th17cytokine profiles levels were determined by MAGPIX Bio-plex assay in supernatants from cell cultures. We observed in supernatants of PBMCs from HS treated with rhMIF a predominance of Th17 cytokine profile with an increase of IL-17A, IL-17F and IL-21 versus PBMCs from SLE patients, which showed an inflammatory profile represented by increase of IL-6 cytokine. According to SLE remission/activity presented at enrollment in the study (Mex-SLEDAI index), the PBMC from active SLE patients showed higher levels of TNF-α and IL-6 versus PBMC from remission SLE patients. In conclusion, our results suggest that MIF can induce a differential inflammatory response in physiological and pathological conditions with a predominance of a Th17 cytokine profile in PBMC from HS and an increase in TNF-α and IL-6 expression in PBMC from active SLE patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, ‎protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: We reported earlier that the cytokine macrophage migration inhibitory factor (MIF) is a potential biomarker in burn injury. In the present study, we investigated the clinical significance of the newly discovered MIF family member d-dopachrome tautomerase (DDT or MIF-2) and their common soluble receptor CD74 (sCD74) in severely burned patients., Methods: DDT and sCD74 serum levels were measured 20 severely burned patients and 20 controls. Serum levels were correlated to the abbreviated burn severity index (ABSI) and total body surface area (TBSA) followed by receiver operating characteristic (ROC) analysis. Data were supported by gene expression dataset analysis of 31 burn patients and 28 healthy controls., Results: CD74 and DDT were increased in burn patients. Furthermore, CD74 and DDT also were elevated in septic non-survivors when compared to survivors. Serum levels of DDT showed a positive correlation with the ABSI and TBSA in the early stage after burn, and the predictive character of DDT was strongest at 24h. Serum levels of CD74 only correlated with the ABSI 5 days after injury., Conclusions: DDT may assist in the monitoring of clinical outcome and prediction of sepsis during the early post-burn period. Soluble CD74 and MIF, by contrast, have limited value as an early predictor of death due to their delayed response to burn., Competing Interests: All authors declare no conflict of interest., (Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.)

For more than 15 years, the tautomerase active site of macrophage migration inhibitory factor (MIF) and its catalytic residue Pro1 have been being targeted for the development of therapeutics that block activation of its cell surface receptor, CD74. Neither the biological role of the MIF catalytic site nor the mechanistic details of CD74 activation are well understood. The inherently unstable structure of CD74 remains the biggest obstacle in structural studies with MIF for understanding the basis of CD74 activation. Using a novel approach, we elucidate the mechanistic details that control activation of CD74 by MIF surface residues and identify structural parameters of inhibitors that reduce CD74 biological activation. We also find that N-terminal mutants located deep in the catalytic site affect surface residues immediately outside the catalytic site, which are responsible for reduction of CD74 activation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is involved in eosinophil biology and in type 2 inflammation, contributing to allergic and helminthic diseases. We hypothesized that MIF participates in the pathogenesis of eosinophilic esophagitis (EoE), an allergic condition characterized by esophageal eosinophilic inflammation. MIF is highly expressed in esophageal mucosa of patients with EoE, compared with gastro-esophageal reflux disease and control patients, where it co-localizes predominantly with eosinophils. In vitro, recombinant MIF promotes human eosinophil chemotaxis, while MIF antagonist and CXCR4 antagonist, AMD3100, revert this effect. In a model of EoE induced by ovalbumin, Mif-deficient mice have reduced inflammation and collagen deposition compared with wild-type (WT) mice. Importantly, treatment of WT mice with anti-MIF or with AMD3100 during the challenge phase prevents accumulation of eosinophils and tissue remodeling. Conversely, recombinant MIF promoted tissue eosinophil inflammation in allergic mice. Together, these results implicate MIF in the pathogenesis of esophageal inflammation and suggest that targeting MIF might represent a novel therapy for EoE.

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Oxidative stress, microglial activation and inflammatory responses seem to contribute to the pathogenesis. The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules. The formation of advanced glycation end products (AGEs), the first ligand of RAGE identified, requires a complex series of reactions including nonenzymatic glycation and free radical reactions involving superoxide-radicals and hydrogen peroxide. Binding of RAGE ligands results in activation of nuclear factor-kappaB (NF-κB). We show that RAGE ablation protected nigral dopaminergic neurons against cell death induced by the neurotoxin MPTP that mimics most features of PD. In RAGE-deficient mice the translocation of the NF-κB subunit p65 to the nucleus, in dopaminergic neurons and glial cells was inhibited suggesting that RAGE involves the activation of NF-κB. The mRNA level of S100, one of the ligands of RAGE, was increased after MPTP treatment. The dopaminergic neurons treated with MPP(+) and S100 protein showed increased levels of apoptotic cell death, which was attenuated in RAGE-deficient mice. Our results suggest that activation of RAGE contributes to MPTP/MPP(+)-induced death of dopaminergic neurons that may be mediated by NF-κB activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Triple-negative breast cancer is difficult to treat because of the lack of rationale-based therapies. There are no established markers and targets that can be used for stratification of patients and targeted therapy. Here we report the identification of novel molecular features, which appear to augment metastasis of triple negative breast tumors. We found that triple-negative breast tumors can be segregated into 2 phenotypes based on their genome-wide protein abundance profiles. The first is characterized by high expression of Stat1, Mx1, and CD74. Seven out of 9 tumors from this group had invaded at least 2 lymph nodes while only 1 out of 10 tumors in group 2 was lymph node positive. In vitro experiments showed that the interferon-induced increase in Stat1 abundance correlates with increased migration and invasion in cultured cells. When CD74 was overexpressed, it increased cell adhesion on matrigel. This effect was accompanied with a marked increase in the membrane expression of beta-catenin, MUC18, plexins, integrins, and other proteins involved in cell adhesion and cancer metastasis. Taken together, our results show that Stat1/CD74 positive triple-negative tumors are more aggressive and suggest an approach for development of better diagnostics and more targeted therapies for triple negative breast cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a "proof-of-concept" for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Fibrocytes were first described in 1994 as fibroblast-like, peripheral blood cells. These bone marrow-derived mesenchymal progenitor cells migrate into regions of tissue injury. They are unique in their expression of hematopoietic and monocyte lineage markers and extracellular matrix proteins. Several studies have focused on the specific role of fibrocytes in the process of wound repair and tissue regeneration. We discuss herein the biology and mechanistic action of fibrocytes in wound healing, scar formation, and maintenance of tissue integrity. Fibrocytes synthesize and secrete different cytokines, chemokines, and growth factors, providing a wound milieu that supports tissue repair. They further promote angiogenesis and contribute to wound closure via pathways involving specific cytokines, leukocyte-specific protein-1, serum amyloid P, and adenosine A(2A) receptors. Fibrocytes are involved in inflammatory fibrotic processes in such diseases as systemic fibrosis, atherosclerosis, asthma, hypertrophic scarring, and keloid formation. Accumulating literature has emphasized the important role of fibrocytes in wound healing and fibrosis. Detailed mechanisms nevertheless remain to be investigated to elucidate the full therapeutic potential of fibrocytes in the treatment of fibrosing disorders and the enhancement of tissue repair., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Psoriasis is a common chronic inflammatory skin disorder with a high prevalence (3-5%) in the Caucasian population. Although the number of capillary vessels increases in psoriatic lesions, there have been few reports that have specifically examined the role of angiogenesis in psoriasis. Angiogenic factors, such as vascular endothelial growth factor (VEGF), may dominate the activity of anti-angiogenic factors and accelerate angiogenesis in psoriatic skin., Objective: We investigated to identify small peptide mimetics of PEDF that might show anti-angiogenic potential for the topical treatment for psoriasis., Methods: We examined the expression of PEDF in skin by immunohistochemical staining, immunoblotting, and RT-PCR. To identify potential PEDF peptides, we screened peptides derived from the proteolytic fragmentation of PEDF for their anti-proliferative action. Anti-psoriatic functions of these peptides were analyzed using a mouse graft model of psoriasis., Results: The specific low-molecular weight peptides (MW<850 Da) penetrated the skin and showed significant anti-angiogenic activity in vitro. Topical application of these peptides in a severe combined immunodeficient mouse model of psoriatic disease led to reduced angiogenesis and epidermal thickness., Conclusions: These data suggest that low-molecular PEDF peptides with anti-angiogenic activity may be a novel therapeutic strategy for psoriasis., (2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Aims: LDL is subjected to glycoxidation in diabetes mellitus. We have evaluated the effect of glycoxidized LDL on lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) expression in endothelial cells in vitro, as well as the relationship between glycoxidzied LDL and LOX-1 in type 2 diabetic patients with and without microalbuminuria in vivo., Methods: Endothelial cells were incubated with modified LDL including glycoxidized LDL, oxidized LDL (oxLDL), glycated LDL, and acetylated LDL, and cellular LOX-1 and the soluble forms of LOX-1 (sLOX-1) in cell medium was measured. Glycoxidized LDL in diabetic patients was determined by measuring the glycoxidation product N(epsilon)-(carboxymethyl)lysine (CML) in apolipoprotein (apo) B. Serum oxLDL and sLOX-1 was determined by ELISA., Results: Only glycoxidized LDL and oxLDL significantly increased LOX-1 expression (p<0.05) and the production of sLOX-1 (p<0.05), and the effect of glycoxidized LDL was greater than that of oxLDL. Both normoalbuminuric (n=110) and microalbuminuric (n=91) patients had higher serum apoB-CML than controls (n=105) (p<0.01), but oxLDL was only elevated in the microalbuminuric patients (p<0.05). Serum sLOX-1 was significantly increased in both groups of patients compared to controls (p<0.01). Serum sLOX-1 correlated with apoB-CML (r=0.36, p<0.001) but not with oxLDL. The relationship between sLOX-1 and apoB-CML was independent of HbA1c, age, gender, BMI and smoking status., Conclusion: Glycoxidized LDL was more potent than oxLDL in inducing LOX-1 in vitro. Serum concentration of apoB-CML, a marker of glycoxidized LDL, was increased in type 2 diabetic patients with and without microalbuminuria, and this was associated with an increase in serum sLOX-1.

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.

Recent experimental and clinical studies have placed new emphasis on the role of the innate immune system in SLE. Nucleic acid-containing immune complexes activate the innate response by engaging specific Toll-like receptors (TLRs) and promote the generation of autoantibodies. Pharmacologic modulation of TLR-directed pathways may offer new therapeutic approaches for the treatment of SLE.

The lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) can be proteolytically cleaved and released as soluble forms (sLOX-1). We have determined serums LOX-1 in type 2 diabetes and evaluated the effect of glucose and advanced glycation end products (AGEs) on sLOX-1 in vitro and in vivo. Endothelial cells were incubated with glucose or AGEs, and sLOX-1 in cell medium was measured. Serum sLOX-1 was measured in 219 diabetic patients and 187 controls by ELISA. The effect of lowering glucose and AGEs on sLOX-1 was determined in 38 poorly controlled diabetic patients after improvement in glycemic control. Incubation of endothelial cells with AGE-BSA led to a dose-dependent increase in sLOX-1, whereas the effect of glucose on sLOX-1 was less marked. Serum sLOX-1 was 9% higher in diabetic patients compared with controls (P<0.01). In the poorly controlled patients, serum sLOX-1 decreased by 12.5% after improvement in glycemic control (P<0.05). The magnitude of reduction in sLOX-1 correlated with the improvement in hemoglobin A1c and AGEs but not with the reduction in oxidized LDL. sLOX-1 level is increased in type 2 diabetes. Both glucose and AGEs are important determinants of LOX-1 expression, and lowering glucose and AGEs leads to a reduction in sLOX-1.

Since the discovery of the circulating fibrocyte as a collagen-producing cell of the peripheral blood, the physiologic and pathologic role of this unique cell population has grown steadily. The present review summarizes the known biology of fibrocytes and discusses evidence for their role in the pathogenesis of nephrogenic systemic fibrosis. Possible mechanisms by which gadolinium may influence the activation or trafficking properties of fibrocytes leading to tissue fibrosis are discussed.

Most mature follicular B cells circulate within the periphery in a quiescent state, without actively contributing to an acute immune response. Lasting B-cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. We recently demonstrated that cell surface CD74 controls mature B-cell survival. Stimulation of cell surface CD74 leads to NF-kappaB activation, which enables entry of the stimulated B cells into the S phase, induction of DNA synthesis, and cell division, and augments the expression of survival genes. In the present study, we investigated CD74 target genes to determine the identities of the molecules whose expression is modulated by CD74, thereby regulating B-cell survival. We report that CD74 activates the p65 member of the NF-kappaB family, which in turn up-regulates the expression of p53-related TAp63 proteins. TAp63 then binds and transactivates the Bcl-2gene and induces the production of Bcl-2 protein, thereby providing the cells with increased survival capacity. Thus, the CD74/NF-kappaB/TAp63 axis defines a novel antiapoptotic pathway in mature B cells, resulting in the shaping of both the B-cell repertoire and the immune response.

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.

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.

Inducible 6-phosphofructo-2-kinase (iPFK-2; PFKFB3) produces fructose-2,6-bisphosphate (F2,6BP), which is a potent allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting step in glycolysis. iPFK-2 functions as an activator of anaerobic glycolysis within the hypoxic microenvironment of growing tumors. The early embryo is challenged similarly since the process of vasculogenesis does not begin until after embryonic day 7. We hypothesized that iPFK-2 expression is essential for the survival of the growing embryo. First, we cloned the mouse homolog of iPFK2 and found that it is abundantly expressed in cortical neurons, epithelial cells, and secretory cells of the choroid plexus, pancreas, and adrenal gland of the adult mouse. Using gene targeting, we then disrupted exons 3-7 of the mouse iPFK2 gene, which encode the substrate binding site. No full-term homozygous iPFK-2(-/-) progeny were produced from 11 F7 iPFK-2(+/-) crosses and no homozygous iPFK-2(-/-) embryos were detected after 8 days of embryogenesis.

Macrophage migration inhibitory factor (MIF) has been shown to participate in both experimental and human atherogenesis. Expression of MMP-9 has been shown to play a role in the instability of atherosclerotic plaque. Thus, we hypothesize that MIF may participate in the destabilization of atherosclerotic plaques by stimulating MMP-9 expression. This hypothesis was investigated by examining the expression of MIF and MMP-9 in human atherosclerotic plaques using two-color immunostaining and by determining the potential role of MIF in the induction of MMP-9 expression in vascular smooth muscle cells (VSMC) and macrophages in vitro. Two-color immunohistochemistry demonstrated that MIF was strongly upregulated by macrophages and VSMCs. This was associated with marked increase in MMP-9 expression in vulnerable atheromatous plaques, but not in the fibrous lesions. Upregulation of MIF and MMP-9 in vulnerable atheromatous plaques was associated with the weakening of fibrous caps. The role of MIF in MMP-9 expression was demonstrated by the ability of MIF to directly induce MMP-9 mRNA and protein expression in macrophages and in VSMCs in a dose and time-dependent manner, which was blocked by a neutralizing MIF antibody. In conclusion, MIF and MMP-9 are markedly upregulated in vulnerable atheromatous plaques. The ability of MIF to induce MMP-9 expression in VSMCs and macrophages suggests that MIF may play a role in the destabilization of human atherosclerotic plaques.

Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries, is required for tumors to grow, invade, and metastasize. Vascular endothelial growth factor (VEGF), a specific mitogen to endothelial cells, is a crucial factor for tumor angiogenesis. In this study, we investigated whether minodronate, a newly developed nitrogen-containing bisphosphonate, could inhibit melanoma growth and improve survival in nude mice by suppressing the VEGF signaling. We found here that minodronate inhibited melanoma growth and improved survival in nude mice by suppressing the tumor-associated angiogenesis and macrophage infiltration. Minodronate completely inhibited the VEGF-induced increase in DNA synthesis and tube formation in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation and Ras activation. Furthermore, minodronate inhibited the VEGF-induced expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in endothelial cells. Minodronate decreased DNA synthesis and increased apoptotic cell death of cultured melanoma cells as well. Our present study suggests that minodronate might suppress melanoma growth and improve survival in nude mice by two independent mechanisms; one is by blocking the VEGF signaling in endothelial cells, and the other is by inducing apoptotic cell death of melanoma. The present study provides a novel potential therapeutic strategy for the treatment of melanoma.

Background: The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells., Methods: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [3H]leucine incorporation., Results: AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT1R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta., Conclusion: The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

Since the original description of circulating fibrocytes in 1994, our knowledge of this unique cell population has grown steadily. While initially described in the context of wound repair, fibrocytes have since been found to participate in granuloma formation, antigen presentation, and various fibrosing disorders. Fibrocytes produce matrix proteins such as vimentin, collagens I and III, and they participate in the remodeling response by secreting matrix metalloproteinases. Fibrocytes also are a rich source of inflammatory cytokines, growth factors, and chemokines that provide important intercellular signals within the context of the local tissue environment. Moreover, fibrocytes express the immunological markers typical of an antigen-presenting cell, and they are fully functional for the presentation of antigen to naïve T cells. Fibrocytes can further differentiate, and they may represent a systemic source of the contractile myofibroblast that appears in many fibrotic lesions. Clinically, there is evidence that patients with hypertrophic scars such as keloids, and those affected by scleroderma and other fibrosing disorders have fibrocytes in their lesions. Recently, a new disease entity called nephrogenic fibrosing dermopathy (NFD) has been described, and the fibrocyte may play an important etiopathogenic role in disease development. Nephrogenic fibrosing dermopathy occurs in patients with renal insufficiency and leads to thickening and hardening of the skin, especially of the extremities. Ongoing research is focusing on the molecular signals that influence fibrocyte migration, proliferation, and function in the context of normal physiology and pathology.

Background: Angiotensin II (Ang II) plays an important role in the development of renal injury through its vasoactive and proinflammatory activities. We investigated whether some of the effects of Ang II could be mediated through the production of macrophage migration inhibitory factor (MIF)., Methods: Groups of rats underwent sham surgery (sham), subtotal nephrectomy (STNx), or STNx plus treatment with irbesartan. Renal tissue was examined 12 weeks postsurgery for MIF mRNA expression and leukocyte accumulation. To determine whether Ang II had a direct effect on MIF production, mRNA synthesis and protein secretion were examined in proximal tubular epithelial (NRK52E and MCT) cell lines., Results: MIF mRNA was strongly expressed in 5.4%+/- 1.1% (mean +/- SD) of cortical tubules of sham-operated rats. This was significantly up-regulated in STNx rats (44.9%+/- 22.6%) and was abrogated by administration of irbesartan (2.8%+/- 2.4%). STNx resulted in significant glomerular and interstitial accumulation of macrophages and T cells, which correlated with glomerular and tubular MIF mRNA expression, respectively. In vitro studies of tubular epithelial cells revealed that Ang II caused a twofold increase in MIF mRNA expression in NRK52E and MCT cells, which was abrogated by irbesartan. In addition, Ang II induced a rapid release of 50% of MIF protein from NRK52E cells within 20 minutes., Conclusion: This study has demonstrated that Ang II up-regulates MIF mRNA production and MIF protein secretion by tubular epithelial cells. Ang II may promote accumulation and activation of interstitial leukocytes via induction of MIF synthesis and secretion in renal tubular epithelial cells. This may be an important mechanism by which Ang II mediates renal injury.

Background: Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine released mainly from macrophages and activated lymphocytes. Both atherosclerosis and abdominal aortic aneurysm (AAA) are inflammatory diseases tightly linked to the function of these cells. The correlation and contribution of MIF to these human diseases remain unknown, although a recent rabbit study showed expression of this cytokine in atherosclerotic lesions., Material and Methods: MIF immunohistochemistry was performed on tissue sections from five normal aortas, seven atherosclerotic carotids, and six AAAs. A group of 112 men with small AAAs (defined as 3 to 5 cm) was recruited at the time of diagnosis, had serum samples taken, and was followed annually for 1 to 5 years (mean, 2.9 years) and referred for surgery if the AAA exceeded 5 cm in diameter. Of this study group, 98 had serum MIF measured with an enzyme-linked immunosorbent assay and 61 had detectable levels., Results: In human atherosclerotic and aneurysmal lesions, MIF protein colocalized in macrophages, endothelial cells, and smooth muscle cells, but normal arteries had negligible MIF expression. Furthermore, serum-MIF levels correlated significantly with annual expansion rate (r = 0.28; P =.005), persisting after adjustment for initial AAA size, smoking habits, diastolic blood pressure, ankle blood pressure index, and age. After exclusion of 38 cases with MIF levels below the detection limit, initial AAA size was also significantly correlated with the MIF levels (r = 0.42; P =.001), persisting after adjustment for similar confounders, and the correlation coefficient with expansion rate increased to 0.42 (P =.001)., Conclusion: Highly expressed MIF in macrophages, endothelial cells, and smooth muscle cells in lesions from atherosclerosis and AAA and significant association between serum MIF level and AAA initial size and AAA expansion rate in a group of patients with AAA suggest a potential involvement of this proinflammatory cytokine in the pathogenesis of these cardiovascular diseases.

Glucocorticoid hormones are important anti-inflammatory agents because of their anti-inflammatory and proapoptotic action within the immune system. Their clinical usefulness remains limited however by side effects that result in part from their growth inhibitory action on sensitive target tissues. The protein mediator, macrophage migration inhibitory factor (MIF), is an important regulator of the host immune response and exhibits both glucocorticoid-antagonistic and growth-regulatory properties. MIF has been shown to contribute significantly to the development of immunopathology in several models of inflammatory disease. Although there is emerging evidence for a functional interaction between MIF and glucocorticoids in vitro, little is known about their reciprocal influence in vivo. We investigated the expression of MIF in rat tissues after ablation of the hypothalamic-pituitary-adrenal axis and after high-dose glucocorticoid administration. MIF expression is constitutive and independent of the influence of adrenal hormones. Hypophysectomy and the attendent loss of pituitary hormones, by contrast, decreased MIF protein content in the adrenal gland. Administration of dexamethasone was found to increase MIF protein expression in those organs that are considered to be sensitive to the growth inhibitory effects of glucocorticoids (immune and endocrine tissues, skin, and muscle). This increase was most likely because of a posttranscriptional regulatory effect because tissue MIF mRNA levels were not influenced by dexamethasone treatment. Finally, MIF immunoneutralization enhanced lymphocyte egress from blood during stress-induced lymphocyte redistribution, consistent with a functional interaction between MIF and glucocorticoids on immune cell trafficking in vivo. These findings suggest a role for MIF in both the homeostatic and physiological action of glucocorticoids in vivo.

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.

Neurofilament (NF)-bound AGEs colocalize immunochemically with SOD1 in the motoneurons of patients with ALS. Among three types of AGE receptors reported in the human brain, AGE-R1 (oligosaccharyltransferase family) and AGE-R2 (substrate of protein kinase C) have been found in neurons, while AGE-R3 is restricted to glia. The present study investigates which of these receptors may be responsible for binding AGEs in the NF conglomerates of motoneurons. Immunostaining of paraffin sections from eight ALS patients (five sporadic and three familial) and three control cases was performed with antibodies directed against R1 and R2, in parallel with those against AGEs and SOD1. The sites of AGE-R1 immunoreactivity (IR) in motoneurons were in conformity to those of NF-associated AGE and SOD1 IRs. By contrast, the IR of R2 was negative in NF conglomerates. Negative R2 IR for NF conglomerates was outlined by surrounding coarse R2 immunopositive granules in the perikaryon. No IR for R1 or R2 was found in hyaline or Bunina inclusions. There was no extraneuronal expression of IR for AGE-R1 or AGEs in microglia or astroglia around the NF accumulation. The colocalization of AGE, AGE-R1, and SOD1 at NF conglomerates in motoneurons supports the notion that AGE-mediated oxidative stress and protein aggregation may be implicated in NF conglomeration and ALS pathogenesis.

Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. The acetylation of core histone proteins is reversible and regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increasing evidence suggests a connection between histone acetylation and the development of cancer and leukemia. We have recently mapped HDAC3 to mouse chromosome 18B3, a region which is syntenic with human chromosome 5q31, where HDAC3 is imbedded in a group of potential tumor suppressor genes and which has been reported to be the smallest commonly deleted segment in malignant myeloid disease. We report herein the identification and characterization of HDAC3, a yeast RPD3 ortholog in the mouse. Studies on murine HDAC3 may yield important insights on the understanding of myeloproliferative disease in humans., (Copyright 1999 Academic Press.)

Advanced glycosylation endproducts (AGEs) which result from the non-enzymatic interaction of proteins and glucose are implicated in the vasculopathy of diabetes and aging. Since aminoguanidine (A) inhibits the accumulation of AGEs, we explored its effects on the development of atherosclerosis. Male New Zealand white cross rabbits fed a high cholesterol (1%) diet were randomized to control (C) or increasing doses of A treatment (25, 50 and 100 mg/kg A body weight). The animals were sacrificed after 12 weeks. Sudan IV was used to stain the lipid containing plaques of the aortic arch, thoracic and abdominal aorta and the surface area occupied by atheroma was assessed. Increasing doses of A treatment were associated with reduction in plaque formation in the aorta. At a dose of 100 mg/kg A, there was a 30, 49 and 48% reduction in plaque formation in the aortic arch, thoracic and abdominal aorta, respectively. There was a correlation between AGE levels and the degree of atheroma in these cholesterol fed rabbits (control, r = 0.75, P < 0.01; 100 mg/kg A, r = 0.59, P = 0.02). These data suggest that advanced glycation may participate in atherogenesis and raise the possibility that inhibitors of advanced glycation may retard this process.

Patients with diabetes and renal insufficiency (Db/ESRD), a group subject to accelerated atherosclerosis exhibit marked increases in the levels of circulating, glycation-derived reactive substances, termed advanced glycation endoproducts (AGEs). These products have been previously shown to react covalently with apoliprotein B (ApoB) to form AGE-ApoB, a modification that results in delayed low density lipoprotein (LDL) clearance and possibly to dyslipidemia. Because the effect of hemodialysis on AGE removal was shown to be unsatisfactory, based on single intradialytic studies, we examined the effect of long-term hemodialysis therapy on serum AGE-ApoB levels, as well as on total serum ApoB of 25 Db/ESRD patients treated by two types of hemodialysis filters, the Fresenius Inc. F8, as the low flux (LF), or high-flux polysulfone AN69 (HF) for two months using an AGE-specific ELISA. At the end of eight weeks, circulating AGE-ApoB levels were reduced significantly (by 35%) from baseline (P = 0.039) in patients treated by HF compared to a modest 16% reduction noted in patients treated by LF (P = 0.05) N = 12, P = 0.047). Of note, total plasma ApoB was reduced by 27% from baseline (P = 0.02) in patients treated by HF compared to a 6% reduction noted in those treated with LF (P = 0.8). In vitro comparison of AGE mass balance, and mass adsorption by the different filters revealed that the higher efficiency of HF filter was due to greater adsorption. The association of reduced AGE-ApoB levels with a decrease in total circulating ApoB by HF and not by LF dialysis suggests: (1) a causal link between AGE clearance and dyslipidemia in diabetic ESRD, and, (2) that more efficient modes of renal replacement treatment and AGE removal could significantly benefit clinical outcome.

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.

Macrophage migration inhibitory factor (MIF) is an important constituent of the host response to stress and infection and is the first mediator that has been identified to be released from immune cells upon stimulation with glucocorticoids. MIF also has been shown to be secreted from the anterior pituitary gland, monocytes/macrophages, and T cells activated by various proinflammatory stimuli. Once released, MIF acts to counter-regulate the inhibitory effect of glucocorticoids on inflammatory cytokine production. To characterize more precisely the role of MIF in the host response to infection, we undertook a systematic analysis of MIF expression in various organs of the rat after endotoxin (lipopolysaccharide) administration. MIF protein and mRNA were analyzed by immunohistochemistry and in situ hybridization, respectively. MIF was found to be expressed constitutively in organs such as the lung, liver, kidney, spleen, adrenal gland, and skin. Significant quantities of MIF protein were detected preformed in various cell types and appeared to be released as a consequence of endotoxemia. In virtually all tissues examined, the loss of MIF protein 6 hours after lipopolysaccharide administration was accompanied by the induction of MIF mRNA and, at 24 hours, by the restoration of immunoreactive, intracellular MIF. The constitutive production of MIF by several cell and tissue types together with its rapid release from intracellular pools distinguishes MIF from other cytokines or hormonal mediators and significantly expands the physiological role of this unique counter-regulator of glucocorticoid action.

Macrophage migration inhibitory factor (MIF), a key mediator of the delayed-type hypersensitivity response, was originally thought to be produced by activated T cells. However, recent studies have found that MIF is produced in many cell types including monocytes/macrophages and anterior pituitary cells. The current study has examined MIF expression in normal and diseased kidney using in situ hybridization, immunohistochemistry, and Northern blotting. MIF mRNA and protein are constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells and some glomerular visceral and parietal epithelial cells. During the development of rat anti-glomerular basement membrane glomerulonephritis, a model of macrophage-mediated renal injury, there was marked de novo expression of MIF by intrinsic kidney cells including endothelium and glomerular and tubular epithelial cells. Up-regulation of MIF expression correlated with macrophage accumulation within the glomerulus (P < 0.001) and tubulointerstitium (P < 0.001). Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, contributing to focal glomerular and tubulointerstitial lesion formation. In addition, up-regulation of MIF expression by parietal epithelial cells was associated with macrophage accumulation within Bowman's space and crescent formation. Combined in situ hybridization and immunostaining also demonstrated MIF expression by macrophages, T cells, and fibroblast-like cells within renal lesions. In conclusion, these data provide the first demonstration that renal epithelial cells are a major source of MIF in both normal and diseased kidney. Furthermore, the up-regulation of MIF expression may play an important role in macrophage accumulation and progressive renal injury in rat crescentic glomerulonephritis.

Much data support a role for both low density lipoprotein (LDL) oxidation and glycation in atherogenesis. While alpha-tocopherol decreases the oxidative susceptibility of LDL, its role in decreasing LDL glycation is unclear. Hence we tested the effect of alpha-tocopherol both in vitro and in vivo on LDL oxidation and glycation. LDL was isolated after enrichment of plasma with alpha-tocopherol. This resulted in a 2-fold increase in alpha-tocopherol in LDL (AT-LDL). During a 6-day incubation of control LDL (C-LDL) and AT-LDL with 25 mM glucose, there were no significant differences in the degree of glycation on days 1, 3, and 6. Also, apoB advanced glycosylation end product levels were not significantly different between C-LDL and AT-LDL. There was a progressive increase in the susceptibility of LDL to oxidation with increasing LDL glycation as evidenced by reduced lag time of copper-catalyzed LDL oxidation. However, AT-LDL was more resistant to copper-catalyzed oxidation. Similar findings were observed when the LDLs were incubated with endothelial cells. The data from the alpha-tocopherol supplementation study confirmed our in vitro findings that alpha-tocopherol significantly decreases oxidative susceptibility of LDL, but does not affect its glycation. Therefore, while glycation increases LDL oxidative susceptibility, alpha-tocopherol decreases the oxidation of glycated LDL but not LDL glycation.

Single, double, and triple immunostaining of cryostat sections of elderly normal and Alzheimer disease (AD) brain was performed with monoclonal and polyclonal antibodies to advanced glycation end products (AGE). The sections were counterstained with thioflavin-S or with immunocytochemistry for A beta and also stained with markers for microglia. AGE-immunoreactivity was detected in senile plaques and neurofibrillary tangles (NFT). AGE immunoreactivity was most intense in dense or reticular amyloid deposits and extracellular NFT, while intracellular NFT and diffuse amyloid had less AGE immunoreactivity. This pattern of immunoreactivity was similar to that noted in previous studies with antibodies to apolipoprotein-E (apo-E). Therefore, double labeling with antibodies to apo-E and AGE was performed. AGE immunoreactivity colocalized to a very high degree with apo-E immunoreactivity, except that relatively more intense apo-E immunoreactivity was detected in amyloid deposits and more intense AGE immunoreactivity in NFT. The lesions that were immunostained with antibodies to AGE and apo-E were often, but not always, associated with a local microglial reaction. The results raise the possibility that apo-E or a fragment of apo-E may be glycated. Biochemical studies are needed to determine the extent of possible apo-E glycation in AD. The present results raise the possibility that glycation may serve as one of the signals for activation of microglia associated with amyloid deposits and extracellular NFT.

It has been postulated that the accumulation of advanced glycation end products (AGEs) in the kidney is important in the pathogenesis of diabetic nephropathy. Previously, aminoguanidine has been shown to inhibit the accumulation of renal AGEs and to retard the development of experimental diabetic nephropathy. The present study serially assessed the accumulation of AGEs in the aorta and kidney, as well as renal functional and structural parameters over 32 weeks of experimental diabetes in the absence and presence of aminoguanidine. In addition, it was determined if aminoguanidine was more effective if administered earlier or later in the evolution of diabetic nephropathy by treating diabetic rats with aminoguanidine in the first or second half of the 32-week study period. In the serial studies, glomerular and renal tubular fluorescence increased over the 32 week period and this increase was attenuated by aminoguanidine treatment. Concomitant with the effects of aminoguanidine on fluorescence, there was a retardation in the rise in urinary albumin excretion and prevention of mesangial expansion. Early or late administration of aminoguanidine in diabetic rats reduced tissue fluorescence in glomeruli and renal tubules. At 32 weeks, renal AGEs were increased in diabetic rats as assessed by tissue fluorescence. Using a specific RIA, renal AGEs were increased in diabetic rats and decreased by aminoguanidine treatment, administered over the entire 32 weeks or in the first or latter half of the 32-week study period. Aminoguanidine therapy for the entire 32-week study period retarded the rise in albuminuria in the diabetic rats and was more effective than 16 weeks of treatment either in the first or second half of the study. Early and late aminoguanidine administration were similar in their capacity to retard the development of albuminuria in diabetic rats. Similar effects were observed on mesangial expansion. The increased glomerular basement thickness in diabetic rats was not affected by aminoguanidine, irrespective of duration or timing of therapy. This study confirms that in vivo generation of AGEs in the kidney is time dependent and closely linked to the development of experimental diabetic nephropathy. The renoprotective effects of aminoguanidine in diabetes appear to be related to the duration but not to the timing of treatment.