Idiopathic detrusor underactivity (DU) and detrusor decompensation which develops following partial bladder outlet obstruction (pBOO) are both associated with smooth muscle degeneration and fibrosis. Macrophage migration inhibitory factor (MIF), an important mediator of bladder inflammation, has been shown to promote fibroblast survival and muscle death in other tissues. We evaluated the hypothesis that MIF has similar actions in the bladder by studying detrusor responses to pBOO or sham surgery in anesthetized female mice rendered null for the mif gene (MIF KO) and in wild-type (WT) controls, all killed 3 wk after surgery. WT mice revealed intense MIF immunoreactivity in urothelial cells which decreased, without change in overall mif mRNA levels. Stereologically sound quantitative morphometric measurements were performed in the middetrusor region of each bladder. MIF KO bladders were normal in appearance, yet were 30-40% heavier, with increased middetrusor collagen and muscle, compared with WT controls. In WT mice, pBOO increased the collagen-to-muscle ratio 1.9-fold and middetrusor collagen 1.8-fold, while nucleated muscle counts were 22% lower. In MIF KO mice, by contrast, pBOO had no significant effect on any of these parameters. In primary bladder muscle cultures, treatment with rMIF protein increased TUNEL staining, raising the proportion of early and late apoptotic cells on flow cytometry. Our studies implicate MIF in the sequence of events leading to detrusor muscle loss and fibrosis in obstruction. They raise the possibility that strategies designed to antagonize MIF synthesis, release, or biological activity could prevent or delay DU and urinary retention.

Glucocorticoid receptors (GRs) are present at a high density in the nucleus of the solitary tract (NTS), an area of the dorsal hindbrain (DHB) that is critical for blood pressure regulation. However, whether these receptors play any role in the regulation of blood pressure is unknown. We tested the hypothesis that glucocorticoids act in the DHB to increase arterial pressure using two experimental strategies. In one approach, we implanted pellets of corticosterone (Cort) or sham pellets onto the DHB over the NTS. Compared with rats with sham pellets, rats with DHB Cort pellets had an increased (P < 0.05) mean arterial pressure (111 +/- 2 vs. 104 +/- 1 mmHg) and heart rate (355 +/- 9 vs. 326 +/- 5 beats/min) after 4 days. In the second approach, we implanted subcutaneous Cort pellets to increase the systemic Cort concentration and then subsequently implanted pellets of the GR antagonist mifepristone (Mif; previously RU-38486) or sham pellets onto the DHB. Two days of DHB Mif treatment reduced (P < 0.05) mean arterial pressure in those rats with elevated plasma Cort levels (118 +/- 2 vs. 108 +/- 1 mmHg for sham vs. Mif DHB pellets). Cort and Mif pellets placed on the dura had no effects on arterial pressure or heart rate, ruling out systemic cardiovascular effects of the steroids. DHB Cort treatment had no effects on plasma Cort concentration or adrenal weight, indicating that the contents of the DHB Cort pellet did not diffuse into the systemic circulation or into the forebrain areas that regulate plasma Cort concentration in concentrations sufficient to produce physiological effects. Immunohistochemistry for the occupied GRs demonstrated that the Cort and Mif from the DHB pellets were delivered to the DHB with minimal diffusion to the ventral hindbrain or forebrain. We conclude that glucocorticoids act in the DHB to increase arterial pressure.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.

Owing to its implication in a range of pathological conditions, including asthma, rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and cancer, the pleiotropic cytokine macrophage migration inhibitory factor (MIF) has been the subject of intensive recent investigation. In the field of dermatology, MIF is believed to be a detrimental factor in diseases such as systemic sclerosis, atopic dermatitis, psoriasis, eczema and UV radiation damage. However, its contribution to other aspects of cutaneous biology is currently unclear. Although its expression in intact skin is well characterized, little is known about MIF's role in cutaneous homoeostasis. However, recent data do identify MIF as a key player in the immune privilege of hair follicles. Similarly, although MIF is rapidly released and its local expression significantly induced upon wounding, its primary role in the ensuing repair process remains a source of contention. MIF has been identified as being a key effector of the beneficial effects of estrogen on wound repair, yet studies employing Mif null mice, recombinant MIF, and neutralizing anti-MIF antibodies have failed to provide a consensus as to whether it benefits or inhibits healing. In fact MIF appears to be able to exert both positive and negative effects, with the cell-specific relevancy of MIF in wound healing still unclear. Thus, if MIF and/or its downstream targets are to be therapeutically useful in the context of cutaneous repair, more needs to be done to establish the nature and mechanism of action of MIF and its receptors in healing wounds.

Human endothelial cells (EC) are typically resistant to the apoptotic effects of stimuli associated with lung disease. The determinants of this resistance remain incompletely understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by human pulmonary artery EC (HPAEC). Its expression increases in response to various death-inducing stimuli, including lipopolysaccharide (LPS). We show here that silencing MIF expression by RNA interference (MIF siRNA) dramatically reduces MIF mRNA expression and the LPS-induced increase in MIF protein levels, thereby sensitizing HPAECs to LPS-induced cell death. Addition of recombinant human MIF (rhMIF) protein prevents the death-sensitizing effect of MIF siRNA. A common mediator of apoptosis resistance in ECs is the death effector domain (DED)-containing protein, FLIP (FLICE-like inhibitory protein). We show that LPS induces a transcription-independent increase in the short isoform of FLIP (FLIP(s)). This increase is blocked by MIF siRNA but restored with the addition of recombinant MIF protein (rHMIF). While FLIP(s) siRNA also sensitizes HPAECs to LPS-induced death, the addition of rhMIF does not affect this sensitization, placing MIF upstream of FLIP(s) in preventing HPAEC death. These studies demonstrate that MIF is an endogenous pro-survival factor in HPAECs and identify a novel mechanism for its role in apoptosis resistance through the regulation of FLIP(s). These results show that MIF can protect vascular endothelial cells from inflammation-associated cell damage.

The cytokine macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of inflammatory and infectious diseases, however its role in HIV-1 infection is unknown. Here we show that HIV-1-infected patients present elevated plasma levels of MIF, that HIV-1-infected peripheral blood mononuclear cells (PBMCs) release a greater amount of MIF, and that the HIV-1 envelope glycoprotein gp120 induces MIF secretion from uninfected PBMCs. The HIV-1 replication in PBMCs declines when these cells are treated with anti-MIF antibodies, and exposure of HIV-1-infected cells to the ABC-transporter inhibitor probenecid results in inhibition of MIF secretion. The addition of recombinant MIF (rhMIF) to HIV-1-infected PBMCs enhances viral replication of CCR5- or CXCR4-tropic HIV-1 isolates. Using a T CD4(+) cell lineage containing an HIV long terminal repeats (LTR)-Luciferase construct, we detected that rhMIF promotes transcription from HIV-1 LTR. Our results show that HIV-1 induces MIF secretion and suggest that MIF influences the HIV-1 biology through activation of HIV-1 LTR.

BACKGROUND

Comparisons between the genomes of the closely related nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal high rates of rearrangement, with a bias towards within-chromosome events. To assess whether this pattern is true of nematodes in general, we have used genome sequence to compare two nematode species that last shared a common ancestor approximately 300 million years ago: the model C. elegans and the filarial parasite Brugia malayi.

RESULTS

An 83 kb region flanking the gene for Bm-mif-1 (macrophage migration inhibitory factor, a B. malayi homolog of a human cytokine) was sequenced. When compared to the complete genome of C. elegans, evidence for conservation of long-range synteny and microsynteny was found. Potential C. elegans orthologs for II of the 12 protein-coding genes predicted in the B. malayi sequence were identified. Ten of these orthologs were located on chromosome I, with eight clustered in a 2.3 Mb region. While several, relatively local, intrachromosomal rearrangements have occurred, the order, composition, and configuration of two gene clusters, each containing three genes, was conserved. Comparison of B. malayi BAC-end genome survey sequence to C. elegans also revealed a bias towards intrachromosome rearrangements.

CONCLUSIONS

We suggest that intrachromosomal rearrangement is a major force driving chromosomal organization in nematodes, but is constrained by the interdigitation of functional elements of neighboring genes.

OBJECTIVE

Elevated macrophage migration inhibitory factor (MIF) has been implicated as a causal mechanism in a number of disease conditions including cardiovascular disease (CVD), diabetes, and cancer. Excess body fat is associated with an increased risk of numerous health conditions including CVD, diabetes, and cancer. To our knowledge, the association between MIF and obesity status and the effect of weight loss on serum MIF concentrations have not been reported. In this study, we examined the effects of participation in a behavior-based weight loss program on MIF concentrations in obese individuals.

METHODS

Study participants were 71 men and women enrolled in The Cooper Institute Weight Management Program. Participants were predominantly female (68%, n=48), middle-aged (46.5+/-9.8 y), and severely obese (BMI=43.0+/-8.6).

METHODS

Plasma MIF concentrations and other standard risk factors were measured before and after participation in a diet and physical activity based weight management program.

RESULTS

The mean follow-up was 8.5+/-3.0 months with an average weight loss of 14.4 kg (P<0.001). The majority of clinical risk factors significantly improved at follow-up. Median levels of plasma MIF concentration were significantly lower at follow-up (median [IQR]; 5.1[3.6-10.3]) compared to baseline (8.4 [4.3-48.8]; P=0.0005). The percentage of participants with plasma MIF concentration>> or =19.5 mg/nl (highest tertile at baseline) decreased from 33.8 to 5.6% (P<0.001). Further, elevated baseline plasma MIF concentration was associated with markers of beta-cell dysfunction and reductions in MIF were associated with improvements in beta-cell function.

CONCLUSIONS

Circulating MIF concentrations are elevated in obese but otherwise healthy individuals; however, this elevation in MIF is not uniform across individuals. In obese individuals with elevated circulating MIF concentrations, participation in physical activity and a dietary-focused weight management program resulted in substantial reduction in MIF.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that participates in the pathogenesis of endotoxemia and experimental crescentic glomerulonephritis. However, very little is known about how MIF production is regulated in disease. We therefore examined whether tumor necrosis factor alpha (TNF-alpha), a known inducer of MIF expression by macrophages in vitro, up-regulates local and systemic MIF expression in a macrophage-mediated rat model of crescentic glomerulonephritis.

METHODS

Anti-glomerular basement membrane (GBM) glomerulonephritis was induced in groups of six primed rats. Animals were treated with 1 mg/kg soluble TNF-alpha receptor (TNFbp) or saline from the time of disease induction until they were killed on Days 1, 7, or 14. Renal MIF expression was assessed by in situ hybridization, immunohistochemistry, and ELISA, and compared with macrophage accumulation and indices of renal damage.

RESULTS

Although TNFbp treatment on Day 1 of the disease had only a partial effect upon the up-regulation of glomerular MIF expression, on Days 7 to 14 it almost completely abrogated the increase in glomerular and interstitial MIF mRNA and protein expression. In addition, TNFbp treatment significantly inhibited MIF secretion by cultured glomeruli and reduced serum MIF levels. The inhibition of renal MIF expression was paralleled by a significant inhibition of glomerular and interstitial macrophage infiltration (p < 0.001 versus saline treated), a significant suppression of renal injury (proteinuria and serum creatinine), and a marked reduction in histologic damage (glomerular hypercellularity, crescent formation, and interstitial fibrosis; all p < 0.01 versus saline treated).

CONCLUSIONS

This study demonstrates for the first time that TNF-alpha up-regulates local MIF expression by both infiltrating macrophages and resident kidney cells in rat crescentic glomerulonephritis. In addition, TNF-alpha regulates systemic MIF production. Thus, TNF-alpha, together with MIF, may play a pathological role in immunologically induced renal disease.

Six R sequences have been described as members of a new family of dispersed repetitive DNA in the mouse genome (Gebhard et al., 1982). Several sequenced regions were extended in the 5' direction (R1, R2 and R6) and two new sequences were determined (R7 and R8). On the basis of our sequence and blot hybridization data it is concluded that the R sequence are adjacent to the so-called small Bam family (Fanning, 1982), which in turn runs into the MIF sequence part (Brown & Piechaczyk, 1983) of the large Bam sequences (Meunier-Rotival et al., 1982). In one of our clones a sequence of 1290 base-pairs comprises MIF, Bam and R sequences in a contiguous arrangement which seems to be characteristic of the long repeat unit of the mouse genome. Several repeat units were found to be truncated within their Bam or R sequence parts. Evidence is also reported for transposition events involving R sequences; for instance of one R sequence (R1) into another (R7). Two R sequences (R1 and R4) have apparently been transposed together with part of the adjacent Bam sequences. Truncation and transposition events may also explain the imbalance of copy numbers within the large repeat unit (25,000 to 50,000 for the small Bam sequences and 100,000 for the R sequences). The spreading of R sequences and other interspersed DNA sequences within the mouse genome may have occurred by transposition events on the DNA level and/or by transcription, retrotranscription and insertion processes.

Human mesenchymal stem cells (MSCs) are capable of repairing pulmonary disorders, but their efficacy is limited by poor engraftment. A strategy is proposed to augment MSC migration to lung tissue by antagonizing macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. Recombinant MIF (85 ng/ml) inhibited in vitro chemokinesis of multipotent MSCs by nearly 50 and 20% for donor preparations with colony-forming efficiencies of 22 +/- 4% and 66 +/- 3%, respectively (P < 0.05). The small-molecule MIF antagonist, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 85 microg/ml), restored MSC migration for all donors to levels found in the absence of MIF. At this concentration, ISO-1 increased migration to conditioned medium from bronchial epithelial cell cultures by>>or=3-fold for all donor MSC preparations (P < 0.05). Transcript levels for the MIF receptor, CD74, in MSCs were independent of colony-forming efficiency. These data suggest that MIF and its antagonists may be relevant to the control of MSC homing and efficacy of stem cell therapies in a variety of clinical scenarios.

Systemic lupus erythematosus (SLE), a chronic multisystem autoimmune disease with a broad range of clinical manifestations, is associated with accelerated atherosclerosis (AT) and increased risk of cardiovascular complications. Relevant factors directly influencing the development of AT comprise immune complex generation, complement activation, and changes in the production and activity of a complex network of cytokines, including type I and II interferons, B lymphocyte stimulator (BLyS), TNFα, IL-6, IL-17 and migration macrophage inhibitor (MIF). Autoantibodies, also responsible for cytokine expression and activation, play a supplementary key role in the development of AT. Genomic and proteomic studies have contributed to the discovery of genes and proteins involved in AT, including some that may be suitable to be used as biomarkers. All that data has allowed the development of new drugs, most of them evaluated in clinical trials: inhibitors of IFN and TNFα, B cell directed therapies, synthetic oligodeoxynucleotides, intravenous immunoglobulin, or statins. The focus of the present paper is to summarize recent evidence showing the role of cytokines in the development of AT in SLE and the rationale, and safety concerns, in the use of combined therapy to prevent AT and cardiovascular disease.

By targeted disruption of the MIF gene, we have established a mouse strain deficient in macrophage (Mphi) migration inhibitory factor (MIF). Despite previous reports indicating an essential role of MIF in endotoxaemia, an injection of lipopolysaccharide (LPS) into the MIF-deficient mice (maintained under specific pathogen-free conditions) caused shock. No significant difference was detected between the MIF-deficient mutant and normal mice in susceptibility to LPS for endotoxaemia or tumour necrosis factor-alpha (TNF-alpha) formation upon LPS injection. Peritoneal Mphi from the two strains produced TNF-alpha in response to LPS with similar dose responses. Dexamethasone suppressed the LPS-induced TNF-alpha response of Mphi, but no difference was detected between the Mphi from the two strains. These results suggest that endogenous MIF has no significant effect on the LPS-induced TNF-alpha production and no effect on suppression of the response by glucocorticoids. Thus, MIF is not crucial for LPS-induced immune responses leading to shock.

Blastoid cell cultures derived from leukocytes of patients in the acute stage of infectious mononucleosis (IM) and harboring Epstein-Barr (EB) virus in at least 1% of the cells were found to possess antigens in their membranes which presently are indistinguishable from those detected in Burkitt's lymphoma (BL) cells by the techniques employed. It was noted that, in the course of IM, antibodies are formed which react in indirect immunofluorescence tests with membrane antigens of live cells from Burkitt tumor lines as well as from IM leukocyte cultures, including an autochthonous line in the case of one patient. Sets of sera from IM patients were tested which included a serum collected weeks to years before onset of illness. In the majority of these the pre-IM serum failed to react in membrane immunofluorescence (MIF) tests with any of several Burkitt tumor cell lines employed, but in some cases the presera reacted with cells of some but not others of the lines. Possible explanations for these discrepant results have been discussed. The antibodies involved in the MIF test are evidently distinct from those responsible for the EBV and heterophile reactions. Maximal MIF activity is attained long after the other two antibodies have reached maximal titers and antibodies to EBV and membrane antigens seem to persist for years whereas the heterophile reaction turns negative within a few months.

BACKGROUND

Micro-immunofluorescence (MIF) is widely used for chlamydia antibody testing (CAT). Recently a species-specific MIF and three enzyme-linked immunosorbent assay (ELISA) tests have been introduced. We compared five commercially available CAT tests, using laparoscopy as a reference, and evaluated whether combinations of tests could improve the predictive value of CAT.

METHODS

In a consecutive cohort of 315 subfertile women, results of the five CAT tests were correlated to findings at laparoscopy. Likelihood and odds ratios (OR) were calculated for single tests and for combinations of tests.

RESULTS

Of the tests evaluated, MIF Labsystems had the best diagnostic performance (OR 15.7), while pELISA Medac (OR 8.2) was the best of the three ELISA tests. Stepwise logistic regression analysis showed that performance of MIF Labsystems could not be improved by adding a second test. Significant cross-reactivity with C. pneumoniae antibodies was found in all tests evaluated, except in pELISA Medac.

CONCLUSIONS

In screening for tubal factor subfertility, MIF Labsystems was superior to the ELISA tests evaluated, and combining two CAT tests did not improve its predictive value. Economic analysis will show whether serial testing by pELISA Medac, and retesting positive samples by MIF Labsystems, is most cost-effective. In CAT, cross-reactivity with C. pneumoniae antibodies is still a major concern.

Malaria, caused by Plasmodium falciparum and related parasites, is responsible for millions of deaths each year, mainly from complications arising from the blood stages of its life cycle. Macrophage migration inhibitory factor (MIF), a protein expressed by the parasite during these stages, has been characterized in mammals as a cytokine involved in a broad spectrum of immune responses. It also possesses two catalytic activities, a tautomerase and an oxidoreductase, though the physiological significance of neither reaction is known. Here, we have determined the crystal structure of MIF from two malaria parasites, Plasmodium falciparum and Plasmodium berghei at 2.2 A and 1.8 A, respectively. The structures have an alpha/beta fold and each reveals a trimer, in agreement with the results of analytical ultracentrifugation. We observed open and closed active sites, these being distinguished by movements of proline-1, the catalytic base in the tautomerase reaction. These states correlate with the covalent modification of cysteine 2 to form a mercaptoethanol adduct, an observation confirmed by mass spectrometry. The Plasmodium MIFs have a different pattern of conserved cysteine residues to the mammalian MIFs and the side chain of Cys58, which is implicated in the oxidoreductase activity, is buried. This observation and the evident redox reactivity of Cys2 suggest quite different oxidoreductase characteristics. Finally, we show in pull-down assays that Plasmodium MIF binds to the cell surface receptor CD74, a known mammalian MIF receptor implying that parasite MIF has the ability to interfere with, or modulate, host MIF activity through a competitive binding mechanism.

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C(high) inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.

Macrophage migration inhibitory factor (MIF) is a multipotent cytokine implicated in the pathogenesis of numerous inflammatory and autoimmune disorders. Since anti-cytokine therapy is considered to be a promising therapeutic strategy, selective targeting of MIF with either anti-MIF antibody or specific chemical MIF inhibitors might offer new therapeutic avenues for these disorders. Considering the unique relationship between MIF and glucocorticoids, therapeutic antagonism of MIF could also represent an effective approach for steroid-sparing therapies in patients with refractory autoimmune diseases.

Macrophage migration inhibitory factor (MIF) is involved in tumorigenesis by facilitating tumor proliferation and evasion of apoptosis; however, its role in tumor immunity is unclear. In this study, we investigated the effect of MIF on the progression of the syngenic, CT26 colon carcinoma and the generation of tumor regulatory T cells (Tregs). The results showed that the tumor growth rate was significantly lower in MIF knockout (MIF(-/-)) mice than in wild-type (MIF(+/+)) mice. Flow cytometric analysis of both spleen and tumor cells revealed that MIF(-/-) mice had significantly lower levels of tumor-associated CD4(+)Tregs than MIF(+/+) mice. The splenic cells of MIF(-/-) mice also showed a decrease in CD8(+)Tregs, which was accompanied by an increase in CD8-induced tumor cytotoxicity. Interestingly, the inducible Treg response in spleen cells to anti-CD3/CD28 plus IL-2 plus TGF-β was greater in MIF(-/-) mice than in MIF(+/+) mice. Spleen cells of MIF(-/-) mice, stimulated with anti-CD3/CD28, produced lower levels of IL-2, but not TGF-β, than those of MIF(+/+) mice, which was recovered by the addition of recombinant MIF. Conversely, a neutralizing anti-MIF Ab blocked anti-CD3-induced IL-2 production by splenocytes of MIF(+/+) mice and suppressed the inducible Treg generation. Moreover, the administration of IL-2 into tumor-bearing MIF(-/-) mice restored the generation of Tregs and tumor growth. Taken together, our data suggest that MIF promotes tumor growth by increasing Treg generation through the modulation of IL-2 production. Thus, anti-MIF treatment might be useful in enhancing the adaptive immune response to colon cancers.

OBJECTIVE

The glycoprotein macrophage migration inhibitory factor (MIF) is a cytokine that has been shown to promote tumor progression and tumor immune escape in ovarian cancer. The present study investigates MIF in uterine cervical cancer.

METHODS

Eighty surgical biopsies (32 cervical dysplasias, 23 in situ carcinomas and 25 invasive carcinomas) of uterine cervical tissue were evaluated immunohistochemically for MIF expression. In uterine cervical cancer cell lines SiHa and CaSki and their respective supernatants, MIF protein expression was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTS

Immunohistochemical analysis shows that MIF is clearly overexpressed on the protein level in invasive cervical cancer compared to cervical dysplasias. MIF overexpression was confirmed by RT-PCR in surgical biopsies of invasive cervical cancer. Western blotting reveals that the MIF protein is overexpressed in SiHA und CaSki cervical cancer cell lines, whereas the ELISA reveals that cervical cancer cells secrete MIF.

CONCLUSIONS

MIF has been shown to promote tumor immune escape mechanisms in other cancer entities, which makes it an interesting target for cancer therapy, given the known significance of immune mechanisms for uterine cervical cancer. The overexpression of MIF on the protein and mRNA level, as well as its secretion by cervical cancer cells points to a critical role of the protein for the pathogenesis of uterine cervical cancer.

BACKGROUND

Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients.

OBJECTIVE

Identification of molecular mediators that contribute to this fibrotic phenotype.

METHODS

Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts.

RESULTS

Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I; (ii) wound healing proteins: gelsolin-like capping protein; (iii) fibrogenetic proteins: mast cell β-tryptase, macrophage migration inhibitory factor (MIF); (iv) antifibrotic proteins: asporin; (v) tumour suppressor proteins: stratifin, galectin-1, maspin; and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS.

CONCLUSIONS

This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.

Momordica charantia (bitter melon) is commonly known as vegetable insulin, but the mechanisms underlying its hypoglycemic effect remain unclear. To address this issue, the effects of bitter melon extracts on postprandial glycemic responses have been investigated in rats. An aqueous extract (AE), methanol fraction (MF) and methanol insoluble fraction (MIF) were prepared from bitter melon. An oral sucrose tolerance test revealed that administration of AE, MF or MIF each significantly suppressed plasma glucose levels at 30 min as compared with the control. In addition, the plasma insulin level at 30 min was also significantly lower after MF administration than in the control in the oral sucrose tolerance test. By contrast, these effects of bitter melon extracts were not observed in the oral glucose tolerance test. In terms of mechanism, bitter melon extracts dose-dependently inhibited the sucrase activity of intestinal mucosa with IC(50) values of 8.3, 3.7 and 12.0 mg/mL for AE, MF and MIF, respectively. The fraction with a molecular weight of less than 1,300 (LT 1,300) obtained from MF inhibited the sucrase activity most strongly in an uncompetitive manner with an IC(50) value of 2.6 mg/mL. Taken together, these results demonstrated that bitter melon suppressed postprandial hyperglycemia by inhibition of alpha-glucosidase activity and that the most beneficial component is present in the LT 1,300 fraction obtained from MF.

OBJECTIVE

Daphnetin was regarded as the mark compound for quality control of Zushima-Pian, a traditional Chinese medicine tablet for treating rheumatoid arthritis (RA). However, no in vivo study on the therapeutic effects of daphnetin for RA has been reported.

METHODS

The adjuvant arthritic (AA) rat model was developed to evaluate the anti-arthritic effects of daphnetin. After immunized with Freund's complete adjuvant (FCA), the rats were treated with daphnetin (2.25 and 4.5mg/kg) for three weeks. We determined the change of secondary paw swelling and the arthritis scores of these tested rats. The severity of arthritis in the knee joints was evaluated by histological assessment of cartilage destruction. The levels of IL-1, TNF-alpha and MIF in the serum were measured by ELISA.

CONCLUSIONS

Our results showed that daphnetin significantly reduced paw swelling and decreased the arthritis scores. The pathological examination demonstrated that articular cartilage degeneration with synovial hyperplasia and inflammatory cells infiltration in AA rats were suppressed by daphnetin. There was significant reduction in production of interleukin-1 (IL-1), tumor necrosis factor (TNF-alpha) and macrophage migration inhibitory factor (MIF) in serum of AA rats treated with daphnetin except the low dose group (2.25mg/kg) on TNF-alpha. In conclusion, we demonstrates that daphnetin is highly effective on preventing and suppressing the development and progression of adjuvant-induced arthritis and provides direct evidences that daphnetin is one of the active principle of Zushima-Pian for treating rheumatoid arthritis.