Macrophage migration inhibitory factor potentiates autoimmune-mediated neuroinflammation.

Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that is associated with clinical worsening and relapses in multiple sclerosis (MS) patients. The mechanism through which MIF promotes MS progression remains undefined. In this study, we identify a critical role for MIF in regulating CNS effector mechanisms necessary for the development of inflammatory pathology in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Despite the ability to generate pathogenic myelin-specific immune responses peripherally, MIF-deficient mice have reduced EAE severity and exhibit less CNS inflammatory pathology, with a greater percentage of resting microglia and fewer infiltrating inflammatory macrophages. We demonstrate that MIF is essential for promoting microglial activation and production of the innate soluble mediators IL-1ß, IL-6, TNF-α, and inducible NO synthase. We propose a novel role for MIF in inducing microglial C/EBP-ß, a transcription factor shown to regulate myeloid cell function and play an important role in neuroinflammation. Intraspinal stereotaxic microinjection of MIF resulted in upregulation of inflammatory mediators in microglia, which was sufficient to restore EAE-mediated inflammatory pathology in MIF-deficient mice. To further implicate a role for MIF, we show that MIF is highly expressed in human active MS lesions. Thus, these results illustrate the ability of MIF to influence the CNS cellular and molecular inflammatory milieu during EAE and point to the therapeutic potential of targeting MIF in MS.

Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells.

Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34(+) cells) display greater E-selectin binding than those obtained from mouse (lin(-)/Sca-1(+)/c-kit(+) [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34(+) and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ~ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ~ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL." E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures.

G-CSF induces E-selectin ligand expression on human myeloid cells.

Clinical use of G-CSF can result in vascular and inflammatory complications. To investigate the molecular basis of these effects, we analyzed the adherence of G-CSF-mobilized human peripheral blood leukocytes (ML) to inflamed (TNF-alpha-stimulated) vascular endothelium. Studies using parallel plate assays under physiologic flow conditions and intravital microscopy in a mouse inflammation model each showed that ML take part in heightened adhesive interactions with endothelium compared to unmobilized (native) blood leukocytes, mediated by markedly increased E-selectin receptor-ligand interactions. Biochemical studies showed that ML express the potent E-selectin ligand HCELL (ref. 8) and another, previously unrecognized approximately 65-kDa E-selectin ligand, and possess enhanced levels of transcripts encoding glycosyltransferases (ST3GalIV, FucT-IV and FucT-VII) conferring glycan modifications associated with E-selectin ligand activity. Enzymatic treatments and physiologic binding assays showed that HCELL and the approximately 65-kDa E-selectin ligand contribute prominently to the observed G-CSF-induced myeloid cell adhesion to inflamed endothelium. Treatment of normal human bone marrow cells with a pharmacokinetically relevant concentration of G-CSF in vitro resulted in increased expression of these two molecules, coincident with increased transcripts encoding pertinent glycosyltransferases and heightened E-selectin binding. These findings provide direct evidence for a role of G-CSF in the induction of E-selectin ligands on myeloid cells, thus providing mechanistic insight into the pathobiology of G-CSF complications.

Multifunctional dendritic polymers in nanomedicine: opportunities and challenges.

Nanotechnology has resulted in materials that have greatly improved the effectiveness of drug delivery because of their ability to control matter on the nanoscale. Advanced forms of nanomedicine have been synthesized for better pharmacokinetics to obtain higher efficacy, less systemic toxicity, and better targeting. These criteria have long been the goal in nanomedicine, in particular, for systemic applications in oncological disorders. Now, the "holy grail" in nanomedicine is to design and synthesize new advanced macromolecular nanocarriers and to translate them from lab to clinic. This review describes the current and future perspectives of nanomedicine with particular emphasis on the clinical targets in cancer and inflammation. The advanced forms of liposomes and polyethylene glycol (PEG) based nanocarriers, as well as dendritic polymer conjugates will be discussed with particular attention paid to designs, synthetic strategies, and chemical pathways. In this critical review, we also report on the current status and perspective of dendritic polymer nanoconjugate platforms (e.g. polyamidoamine dendrimers and dendritic polyglycerols) for cellular localization and targeting of specific tissues (192 references).

Vitamin E is a MIF Inhibitor.

Macrophage migration inhibitory factor (MIF) is known to contribute to the pathogenesis of inflammatory hyperalgesia and neuropathic pain. Prior studies have shown that Vitamin E treatment is associated with attenuated hyperalgesia and reduced neuropathic pain in rodents. Given these observations, we investigated the possibility that Vitamin E is a MIF inhibitor. Dopachrome tautomerase assays revealed that Vitamin E inhibits the enzymatic activity of purified human recombinant MIF (rhMIF) in a dose-dependent manner (45%, 74%, 92% and 100% inhibition at 3, 10, 30 and 100µM, respectively). Cell-free ELISA based assays showed that Vitamin E binds onto rhMIF thereby blocking its recognition (48% inhibition at 100µM). Circular dichroism studies indicated the Vitamin E has a strong affinity to bind to rhMIF (binding constant 19.52±1.4µM). In silico studies demonstrated that Vitamin E docks well in the active site of MIF with the long aliphatic chain of Vitamin E exhibiting strong van der Waals interactions with MIF. Most importantly, human cell-based assays revealed that Vitamin E significantly inhibits rhMIF-induced production of pro-inflammatory cytokines in a dose-dependent manner (77%, 80%, and 96% inhibition of IL-6 production, respectively, at 10, 30 and 100µM). Taken together, these results demonstrate that Vitamin E inhibits not only the enzymatic activity of MIF but more importantly the biological function of MIF. Our findings suggest that Vitamin E may be attenuating hyperalgesia and reducing neuropathic pain at least in part by inhibiting MIF activity.

Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock*.

BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.

Synthesis and biological activity of novel MIF antagonists.

Two series of novel furan and indole compounds were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds from both series inhibited the enzymatic activity of MIF at levels equal to or significantly better than ISO-1 (an early MIF inhibitor). The majority of the compounds that robustly inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells were from the furan series (compounds 5, 9, 13, 15, and 16). In contrast, compounds that markedly inhibited the MIF-induced production of pro-inflammatory cytokines were predominantly from the indole series (compounds 26, 29, and 32).

Phenotypic and genotypic assessment of concomitant drug-induced toxic effects in liver, kidney and blood.

Several studies have characterized drug-induced toxicity in liver and kidney. However, the majority of these studies have been performed with 'individual' organs in isolation. Separately, little is known about the role of whole blood as a surrogate tissue in drug-induced toxicity. Accordingly, we investigated the 'concurrent' response of liver, kidney and whole blood during a toxic assault. Rats were acutely treated with therapeutics (acetaminophen, rosiglitazone, fluconazole, isoniazid, cyclophosphamide, amphotericin B, gentamicin and cisplatin) reported for their liver and/or kidney toxicity. Changes in clinical chemistry parameters (e.g. AST, urea) and/or observed microscopic tissue damage confirmed induced hepatotoxicity and/or nephrotoxicity by all drugs. Drug-induced toxicity was not confined to an 'individual' organ. Not all drugs elicited significant alterations in phenotypic parameters of toxicity (e.g. ALT, creatinine). Accordingly, the transcriptional profile of the organs was studied using a toxicity panel of 30 genes derived from literature. Each of the test drugs generated specific gene expression patterns which were unique for all three organs. Hierarchical cluster analyses of purported hepatotoxicants and nephrotoxicants each led to characteristic 'fingerprints' (e.g. decrease in Cyp3a1 indicative of hepatotoxicity; increase in Spp1 and decrease in Gstp1 indicative of nephrotoxicity). In whole blood cells, a set of genes was derived which closely correlated with individual drug-induced concomitant changes in liver or kidney. Collectively, these data demonstrate drug-induced multi-organ toxicity. Furthermore, our findings underscore the importance of transcriptional profiling during inadequate phenotypic anchorage and suggest that whole blood may be judiciously used as a surrogate for drug-induced extra-hematological organ toxicity.

A preferential p110alpha/gamma PI3K inhibitor attenuates experimental inflammation by suppressing the production of proinflammatory mediators in a NF-kappaB-dependent manner.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the increased production and/or biological activity of proinflammatory cytokines (e.g., TNF-alpha, IL-6). The production of proinflammatory cytokines is controlled at the gene level by the activity of transcription factors, such as NF-kappaB. Phosphatidylinositol 3-kinase (PI3K), a lipid kinase, is known to induce the activation of NF-kappaB. Given this, we hypothesized that inhibitors of PI3K activation would demonstrate anti-inflammatory potential. Accordingly, we studied the effects of a preferential p110alpha/gamma PI3K inhibitor (compound 8C; PIK-75) in inflammation-based assays. Mechanism-based assays utilizing human cells revealed that PIK-75-mediated inhibition of PI3K activation is associated with dramatic suppression of downstream signaling events, including AKT phosphorylation, IKK activation, and NF-kappaB transcription. Cell-based assays revealed that PIK-75 potently and dose dependently inhibits in vitro and in vivo production of TNF-alpha and IL-6, diminishes the induced expression of human endothelial cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and blocks human monocyte-endothelial cell adhesion. Most importantly, PIK-75, when administered orally in a therapeutic regimen, significantly suppresses the macroscopic and histological abnormalities associated with dextran sulfate sodium-induced murine colitis. The efficacy of PIK-75 in attenuating experimental inflammation is mediated, at least in part, due to the downregulation of pertinent inflammatory mediators in the colon. Collectively, these results provide first evidence that PIK-75 possesses anti-inflammatory potential. Given that PIK-75 is known to exhibit anti-cancer activity, the findings from this study thus reinforce the cross-therapeutic functionality of potential drugs.

Novel derivatives of ISO-1 as potent inhibitors of MIF biological function.

A series of novel 1,2,4-oxadiazole, phthalimide, amide and other derivatives of ISO-1 were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds inhibited MIF enzymatic activity at levels better than ISO-1. Of note, compounds 7, 22, 23, 24, 25 and 27 inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells and, more importantly, inhibited the MIF-induced production of interleukin-6 (IL-6) and/or interleukin-1beta (IL-1beta) significantly better than ISO-1.

Synthesis and therapeutic evaluation of pyridyl based novel mTOR inhibitors.

A series of novel cyanopyridyl based molecules (1-14) were designed, synthesized and probed for inhibition of mammalian target of rapamycin (mTOR) activity. Compound 14 was found to be a potent inhibitor of mTOR activity as assessed by enzyme-linked immunoassays and Western blot analysis. Most importantly, systemic application (intraperitoneal; ip) of compound 14 significantly suppressed macroscopic and histological abnormalities associated with chemically-induced murine colitis.

NF-κB dependent anti-inflammatory activity of chlorojanerin isolated from Saussurea heteromalla.

Medicinal plants have shown great promise as a source of novel drug compounds for the treatment of inflammatory disorders. In our search for new entities with anti-inflammatory potential, the extracts of the whole plant of Saussurea heteromalla (family-Asteraceae), collected from Himalayas, were evaluated in the high throughput screen for TNF-α and IL-6 inhibitors. The extract blocked TNF-α and IL-6 production in LPS stimulated THP-1 cells (human acute monocyte leukemia cell line) completely at 10 and 30 µg/ml. The plant has been found as a new source of chlorojanerin, a guaianolide type of sesquiterpene lactone. Chlorojanerin was shown to be significantly effective in inhibiting TNF-α and IL-6 production in LPS-stimulated THP-1 cells (IC(50)=2.3±0.2 µM and 1.8±0.7 µM respectively). The compound also blocked TNF-α and IL-6 production from LPS-stimulated human monocytes (IC(50)=1.5±0.4 and 0.7±0.2 µM respectively) and synovial cells from a patient with rheumatoid arthritis (IC(50)<0.03 and 0.5 µM respectively). Transcriptional profiling of the LPS stimulated THP-1 cells revealed that chlorojanerin exerted its anti-inflammatory effect by inhibiting the expression of 8 genes involved in activating the transcription factor - NF-κB. Real time analysis of these genes validated the effect of chlorojanerin on the classical downstream targets of NF-κB. Thus, this study clearly delineated 8 genes which were specifically mitigated due to the effect of chlorojanerin on NF-κB induced signaling at the mRNA level. Further, chlorojanerin at 5 µM also inhibited the binding of NF-κB in a GFP reporter assay system by 55.5% thus validating the microarray gene expression data. This work is a step towards the isolation and characterization of lead anti-inflammatory agents from the extract of Saussurea heteromalla, which can be developed into better therapeutic molecules targeted towards some specific inflammatory diseases.

A TSHr-LH/CGr chimera that measures functional TSAb in Graves' disease.

CONTEXT: Stimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves' disease. In a patient's serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time. OBJECTIVE: The objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves' disease. DESIGN: A TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay. SETTING: The study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology). PATIENTS: The assay was validated using sera from 170 individuals with Graves' disease, Hashimoto's thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves' disease) from the University of Pisa. INTERVENTIONS: Interventions included the assessment of patients' sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays. MAIN OUTCOME MEASURES: The Mc4 assay has the best accuracy for diagnosing Graves' disease. RESULTS: The Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively. CONCLUSION: The Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves' disease.

Macrophage migration inhibitory factor (MIF) is essential for inflammatory and neuropathic pain and enhances pain in response to stress.

Stress and glucocorticoids exacerbate pain via undefined mechanisms. Macrophage migration inhibitory factor (MIF) is a constitutively expressed protein that is secreted to maintain immune function when glucocorticoids are elevated by trauma or stress. Here we show that MIF is essential for the development of neuropathic and inflammatory pain, and for stress-induced enhancement of neuropathic pain. Mif null mutant mice fail to develop pain-like behaviors in response to inflammatory stimuli or nerve injury. Pharmacological inhibition of MIF attenuates pain-like behaviors caused by nerve injury and prevents sensitization of these behaviors by stress. Conversely, injection of recombinant MIF into naïve mice produces dose-dependent mechanical sensitivity that is exacerbated by stress. MIF elicits pro-inflammatory signaling in microglia and activates sensory neurons, mechanisms that underlie pain. These data implicate MIF as a key regulator of pain and provide a mechanism whereby stressors exacerbate pain. MIF inhibitors warrant clinical investigation for the treatment of chronic pain.

A TSHR-LH/CGR chimera that measures functional thyroid-stimulating autoantibodies (TSAb) can predict remission or recurrence in Graves' patients undergoing antithyroid drug (ATD) treatment.

CONTEXT: A functional thyroid-stimulating autoantibodies (TSAb) assay using a thyroid-stimulating hormone receptor chimera (Mc4) appears to be clinically more useful than the commonly used assay, a binding assay that measures all the antibodies binding to the thyroid-stimulating hormone receptor without functional discrimination, in diagnosing patient with Graves' disease (GD). OBJECTIVE: The objective of the study was to investigate whether an Mc4 assay can predict relapse/remission of hyperthyroidism after antithyroid drug (ATD) treatment in patients with GD. DESIGN: An Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period. SETTING AND PATIENTS: GD patients from the Chieti University participated in this study. INTERVENTIONS: Interventions included the assessment of patients' sera using the Mc4 assay, the Mc4-derivative assay (Thyretain), and a human monoclonal thyroid-stimulating hormone receptor antibody, M22 assay. MAIN OUTCOME MEASURES: The Mc4 assay, a sensitive index of remission and recurrence, was used in this study. RESULTS: The TSAb levels significantly decreased only in the remitting group as evidenced by Mc4 assay values at the end of ATD (0.96 ± 1.47, 10.9 ± 26.6. and 24.7 ± 37.5 arbitrary units for the remitting, relapsing, and unsuspended therapy groups, respectively). Additional prognostic help was obtained by thyroid volume measurements at the end of treatment. Although not statistically significant, the Mc4 assay has a trend toward improved positive predictive value (95.4 vs. 84.2 or 87.5%), specificity (96.4 vs. 86.4 and 90.9%), and accuracy (87.3 vs. 83.3 and 80.9%) comparing the Mc4, Thyretain, and M22 assays, respectively. Thyretain has a trend toward improved negative predictive value (82.6 vs. 81.8 and 76.9%) and sensitivity (80 vs. 77.8 and 70%) comparing Thyretain, Mc4, and M22 assays, respectively. CONCLUSION: The Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.

7-hydroxyfrullanolide, a sesquiterpene lactone, inhibits pro-inflammatory cytokine production from immune cells and is orally efficacious in animal models of inflammation.

A promising therapeutic approach to reduce pathological inflammation is to inhibit the increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6). In this study, we investigated the anti-inflammatory potential of 7-hydroxyfrullanolide (7HF). 7HF is an orally bioavailable, small molecule sesquiterpene lactone isolated from the fruit of Sphaeranthus indicus. 7HF significantly and dose-dependently diminished induced and spontaneous production of TNF-alpha and IL-6 from freshly isolated human mononuclear cells, synovial tissue cells isolated from patients with active rheumatoid arthritis and BALB/c mice. Oral administration of 7HF significantly protected C57BL/6J mice against endotoxin-mediated lethality. In the dextran sulfate sodium (DSS) model of murine colitis, oral administration of 7HF prevented DSS-induced weight loss, attenuated rectal bleeding, improved disease activity index and diminished shortening of the colon of C57BL/6J mice. Histological analyses of colonic tissues revealed that 7HF attenuated DSS-induced colonic edema, leukocyte infiltration in the colonic mucosa and afforded significant protection against DSS-induced crypt damage. 7HF was also significantly efficacious in attenuating carrageenan-induced paw edema in Wistar rats after oral administration. In the collagen-induced arthritis in DBA/1J mice, 7HF significantly reduced disease associated increases in articular index and paw thickness, protected against bone erosion and joint space narrowing and prominently diminished joint destruction, hyperproliferative pannus formation and infiltration of inflammatory cells. Collectively, these results provide evidence that 7HF-mediated inhibition of pro-inflammatory cytokines functionally results in marked protection in experimental models of acute and chronic inflammation.

Phenyl methimazole suppresses dextran sulfate sodium-induced murine colitis.

Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

A fluorinated analog of ISO-1 blocks the recognition and biological function of MIF and is orally efficacious in a murine model of colitis.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the synthesis and/or biological activity of macrophage migration inhibitory factor (MIF). Prior studies have shown that intraperitoneal administration of small-molecule inhibitors targeting the catalytic pocket of MIF (e.g., ISO-1) elicits a therapeutic effect in mouse inflammation models. However, it remains to be elucidated whether these tautomerase activity inhibitors block the synthesis and/or biological activity of MIF. In this study, we investigated and compared the activity of representative MIF inhibitors from isoxazole series (fluorinated analog of ISO-1; ISO-F) and substituted quinoline series (compound 7E; 7E). Our results demonstrate that ISO-F is a more potent MIF inhibitor than 7E. Both ISO-F and 7E do not inhibit MIF synthesis but "bind-onto" MIF thereby blocking its recognition. However, in contrast to 7E, ISO-F docks well in the active site of MIF and also has a stronger binding affinity towards MIF. In line with these observations, ISO-F, but not 7E, robustly inhibits the biological function of MIF. Most importantly, ISO-F, when administered orally in a therapeutic regimen, significantly suppresses dextran sulphate sodium (DSS)-induced murine colitis. This study, which provides mechanistic insights into the anti-inflammatory efficacy of ISO-F, is the first documented report of in vivo anti-inflammatory efficacy of a MIF inhibitor upon oral administration. Moreover, the findings from this study reinforce the potential of catalytic site of MIF as a target for eliciting therapeutic effect in inflammatory disorders. Compounds (e.g., ISO-F) that block not only the recognition but also the biological function of MIF are potentially attractive for reducing pathological inflammation.

L-selectin-mediated lymphocyte-cancer cell interactions under low fluid shear conditions.

Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07-0.08 dynes/cm(2), consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow.

Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone.

The capacity to direct migration ('homing') of blood-borne cells to a predetermined anatomic compartment is vital to stem cell-based tissue engineering and other adoptive cellular therapies. Although multipotent mesenchymal stromal cells (MSCs, also termed 'mesenchymal stem cells') hold the potential for curing generalized skeletal diseases, their clinical effectiveness is constrained by the poor osteotropism of infused MSCs (refs. 1-3). Cellular recruitment to bone occurs within specialized marrow vessels that constitutively express vascular E-selectin, a lectin that recognizes sialofucosylated determinants on its various ligands. We show here that human MSCs do not express E-selectin ligands, but express a CD44 glycoform bearing alpha-2,3-sialyl modifications. Using an alpha-1,3-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, we converted the native CD44 glycoform on MSCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL), which conferred potent E-selectin binding without effects on cell viability or multipotency. Real-time intravital microscopy in immunocompromised (NOD/SCID) mice showed that intravenously infused HCELL(+) MSCs infiltrated marrow within hours of infusion, with ensuing rare foci of endosteally localized cells and human osteoid generation. These findings establish that the HCELL glycoform of CD44 confers tropism to bone and unveil a readily translatable roadmap for programming cellular trafficking by chemical engineering of glycans on a distinct membrane glycoprotein.