BACKGROUND

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. It contributes toward an exaggerated gram-negative inflammatory response via its ability to induce Toll-like receptor-4 expression. Studies have shown that MIF knockout mice have less aggressive Pseudomonas infection (compared with wild-type).

OBJECTIVE

To assess whether a novel functional MIF polymorphism was associated with clinical prognosis in a patient cohort with chronic gram-negative infection, namely cystic fibrosis (CF).

METHODS

Collected genomic DNA was analyzed via polymerase chain reaction amplification for the polymorphic region for the CATT repeat polymorphism. Individuals may have a 5-, 6-, 7-, or 8-CATT tetranucleotide repeat unit on each allele. The 5-CATT repeat allele exhibits the lowest MIF promoter activity.

RESULTS

Patients with stable CF (n = 167) and a matched control group (n = 166) were enrolled. In patients with CF, the <em>MIF</em>5(+) group had a decreased incidence of Pseudomonas aeruginosa colonization (odds ratio, 0.25; 95% confidence interval, 0.09-0.65; p = 0.004) and a significant reduction in the risk of pancreatic insufficiency (odds ratio, 0.27; 95% confidence interval, 0.07-1.0; p = 0.05). A trend toward milder disease activity in the <em>MIF</em>5(+) group was seen with all other parameters.

CONCLUSIONS

The results support the concept of a regulatory role for MIF in CF.

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.

That endogenous sex steroid hormones profoundly influence the response to cutaneous injury is well established. How they and other factors combine to direct repair in male and female animals is much less well understood. Using a murine incisional wound-healing model, we investigated the roles of circulating sex steroids, macrophage migration inhibitory factor (MIF) (the mediator of delayed healing in ovariectomized animals), and hormone- and MIF-independent factors in controlling repair. We report that d 3 wounds, of comparable size in intact male and female mice, are significantly larger in ovariectomized female animals than in castrated males, suggesting that native sex hormones mask inherent underlying differences in the ways in which males and females respond to wounding. Wound MIF levels were comparable in intact male and female mice but greater in ovariectomized females than castrated males. Furthermore, wound levels of Jun activation domain-binding protein 1 (JAB1), a key factor by which MIF activates intracellular responses, were increased through ovariectomy and greater in ovariectomized females than castrated males. This difference in wound JAB1 levels may underscore the marked sex difference we observed in the responses of MIF knockout mice to the local application of MIF: healing was impaired in ovariectomized females but not castrated males. Separately, systemic treatment with androgens and estrogens yielded contrasting effects on repair in male and female animals. Collectively, the presented data indicate sex divergence in wound healing to be multifaceted, being strongly influenced by MIF and seemingly limited by the combined actions of gonadal steroids.

Inflammatory immune response plays a key role in reproductive failures such as multiple implantation failures (MIF), early pregnancy loss, and recurrent pregnancy losses (RPL). Cellular immune responses particularly mediated by natural killer (NK), and T cells are often dysregulated in these conditions. Excessive or inappropriate recruitment of peripheral blood NK cells to the uterus may lead to cytotoxic environment in utero, in which proliferation and differentiation of trophoblast is hampered. In addition, inadequate angiogenesis by uterine NK cells often leads to abnormal vascular development and blood flow patterns, which, in turn, leads to increased oxidative stress or ischemic changes in the invading trophoblast. T-cell abnormalities with increased Th1 and Th17 immunity, and decreased Th2 and T regulatory immune responses may play important roles in RPL and MIF. A possible role of stress in inflammatory immune response is also reviewed.

Using a mouse model of neonatal respiratory distress syndrome (RDS), we demonstrate a central role for macrophage migration inhibitory factor (MIF) in lung maturation at the developmental stage when human neonates are most susceptible to RDS. We prematurely delivered mouse pups at embryonic day 18, during the early saccular stage of pulmonary development. Only 8% of the prematurely delivered pups genetically deficient in MIF survived 8 h vs 75% of wild-type controls (p<0.001). This phenotype was corrected when pups of all genotypes were bred from dams heterozygote for MIF deficiency. Local production of MIF in the lung increased at embryonic day 18, continued until full-term at embryonic day 19.5, and decreased in adulthood, thus coinciding with this developmental window. The lungs of pups genetically deficient in MIF were less mature upon histological evaluation, and demonstrated lower levels of vascular endothelial growth factor and corticosterone--two factors that promote fetal lung maturation. In vitro studies support a role for MIF in surfactant production by pulmonary epithelial cells. In a cohort of human neonates with RDS, higher intrapulmonary MIF levels were associated with a lower likelihood of developing bronchopulmonary dysplasia, a sequelae of RDS (p<0.03). This study demonstrates for the first time a role for MIF in lung maturation, and supports a protective role for MIF in newborn lung disease.

The diverse actions of macrophage migration inhibitory factor (MIF) within the immuno-neuroendocrine system are yet to be fully understood, but it is clear that MIF plays a pivotal role in the regulation of both the innate and adaptive immune response. An emerging body of data presently indicates that MIF's position within the cytokine cascade is to act in concert with glucocorticoids to control the 'set point' and magnitude of the immune and inflammatory response. In this article we will review the actions of MIF within the immune system and discuss the overlapping and contrasting aspects of MIF and glucocorticoid biology. In particular we will focus on the role of MIF within the immuno-neuroendocrine interface and suggest molecular mechanisms by which MIF may counter-regulate glucocorticoid function. Finally we will discuss emerging evidence that functional MIF gene-promoter polymorphisms render one susceptible to elevated MIF expression, and the development of an exaggerated immune/inflammatory response that potentiates the progression to chronic inflammatory disease.

OBJECTIVE

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE(-/-) mouse.

RESULTS

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE(-/-) mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93(-) CD19(+) B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19(+) B cells, CD4(+) and CD8(+) T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE(-/-) mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93(-) CD19(+) B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

CONCLUSIONS

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE(-/-) mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.

Macrophage Migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine expressed by a variety of cell types. Although MIF has been primarily studied for its role in the pathogenesis of autoimmune diseases, it has also been shown to promote tumorigenesis and it is over expressed in various malignant tumors. MIF is able to induce angiogenesis, cell cycle progression, and to block apoptosis. As tailored therapeutic approaches for the inhibition of endogenous MIF are being developed, it is important to evaluate the role of MIF in individual neoplastic conditions that may benefit from specific MIF inhibitors. Along with this line, in this paper, we have reviewed the evidence of the involvement of MIF in the etiopathogenesis and progression of glioblastoma and the preclinical data suggesting the possible use of specific MIF inhibition as a potential novel therapeutic strategy for brain tumors.

The cellular response to stress involves the recruitment and coordination of molecular signaling pathways that prevent cell death. D-dopachrome tautomerase (DDT) is an enzyme that lacks physiologic substrates in mammalian cells, but shares partial sequence and structural homology with macrophage migration inhibitory factor (MIF). Here, we observed that DDT is highly expressed in murine cardiomyocytes and secreted by the heart after ischemic stress. Antibody-dependent neutralization of secreted DDT exacerbated both ischemia-induced cardiac contractile dysfunction and necrosis. We generated cardiomyocyte-specific DDT knockout mice (Myh6-Cre Ddtfl/fl), which demonstrated normal baseline cardiac size and function, but had an impaired physiologic response to ischemia-reperfusion. Hearts from Myh6-Cre Ddtfl/fl mice exhibited more necrosis and LV contractile dysfunction than control hearts after coronary artery ligation and reperfusion. Furthermore, treatment with DDT protected isolated hearts against injury and contractile dysfunction after ischemia-reperfusion. The protective effect of DDT required activation of the metabolic stress enzyme AMP-activated protein kinase (AMPK), which was mediated by a CD74/CaMKK2-dependent mechanism. Together, our data indicate that cardiomyocyte secretion of DDT has important autocrine/paracrine effects during ischemia-reperfusion that protect the heart against injury.

Hypoxic culture has been shown to delay premature senescence occurring during in vitro culture. Human mesenchymal stem cells (hMSCs) cultured under hypoxia have been reported to maintain their stemness properties and delay senescence compared to the cells cultured under normoxia. However, the molecular mechanism by which hypoxia regulates premature senescence has not been fully revealed. In this study, hMSCs were cultured under the conditions of 21% (normoxia) and 1% O2 (hypoxia) tension and analyzed for cell growth, expression of MSC surface markers, multilineage differentiation, and cellular senescence. Our results showed that more cells retained MSC surface markers in hypoxic culture than those in normoxic culture, and hypoxia was able to enhance multilineage differentiation of hMSCs. The hypoxic condition also delayed cellular senescence of hMSCs, increased activation of AKT signaling, and upregulated both intra- and extracellular levels of macrophage migration inhibitory factor (MIF) compared to the normoxic condition. Inhibition of AKT activity in hypoxic culture increased the number of cells with positive staining for senescence-associated β-galactosidase activity, upregulated expression levels of senescence-associated markers p16 and p21 mRNA transcripts, and decreased expression levels of potency-associated markers, including NANOG, OCT3/4, and SOX2. On the other hand, upregulated intra- and extracellular levels of MIF by stable MIF overexpression in normoxic culture increased the activation of AKT while decreasing mRNA expression of senescence-associated markers and increasing expression of potency-associated markers. Taken together, our findings suggest that hMSCs in hypoxic culture produce endogenous MIF to activate AKT signaling to delay the progression of cellular senescence.

Autoimmunity and chronic low-grade inflammation are hallmarks of diabetes mellitus type one (T1DM) and type two (T2DM), respectively. Both processes are orchestrated by inflammatory cytokines, including the macrophage migration inhibitory factor (MIF). To date, MIF has been implicated in both types of diabetes; therefore, understanding the role of MIF could affect our understanding of the autoimmune or inflammatory responses that influence diabetic pathology. This review highlights our current knowledge about the involvement of MIF in both types of diabetes in the clinical environment and in experimental disease models.

Long non-coding RNAs (lncRNAs) are implicated to be involved in the pathogenesis of many cancers. Herein we report on our discovery of a novel lncRNA, ZFPM2 antisense RNA 1 (ZFPM2-AS1), and its critical role in gastric carcinogenesis. ZFPM2-AS1 expression in gastric cancer specimens was analyzed using Gene Expression Omnibus data set and validated in 73 paired gastric tumor and normal adjacent gastric tissue specimens using qRT-PCR. The effect of ZFPM2-AS1 expression on proliferation and apoptosis in gastric cancer cells was assessed by altering its expression in vitro and in vivo. Mechanistic investigation was carried out using cell and molecular biological approaches. ZFPM2-AS1 expression was higher in gastric tumors than in normal gastric tissue. Also, increased ZFPM2-AS1 expression in gastric cancer specimens was associated with tumor size, depth of tumor invasion, differentiation grade, and TNM stage. High ZFPM2-AS1 expression predicted markedly reduced overall and disease-free survival in gastric cancer patients. Functional experiments demonstrated that ZFPM2-AS1 expression promoted proliferation and suppressed apoptosis of gastric cancer cells in vitro and promoted tumor growth in vivo. This effect is associated with attenuated nuclear translocation of p53. Mechanistic experiments demonstrated that tumor-activated ZFPM2-AS1 could bind to and protect the degradation of macrophage migration inhibitory factor (MIF), a potent destabilizer of p53. Knockdown of MIF expression diminished ZFPM2-AS1's impact on p53 expression in gastric cancer cells. Our findings demonstrated that ZFPM2-AS1 regulates gastric cancer progression and revealed a novel ZFPM2-AS1/MIF/p53 signaling axis, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant gastric cells.

Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

Bone metastasis is an incurable complication of breast cancer affecting 70-80 % of advanced patients. It is a multistep process that includes tumour cell mobilisation, intravasation, survival in the circulation, extravasation, migration and proliferation in the bone marrow/bone. Although novel findings demonstrate the bone marrow microenvironment significance in bone metastatic progression, a majority of studies have focused on end-stage disease and little is known about how the pre-metastatic niche arises in the bone marrow/bone tissues. We demonstrated a significant increase in patients' peripheral blood plasma ability to induce transendothelial migration of MCF-7 cells compared with healthy volunteers. Moreover, high RANKL, MIF and OPG levels in patients' peripheral blood could play a role in the intravasation, angiogenesis, survival and epithelial-mesenchymal transition of circulating tumour cells. Also, we observed a significant increase in patients' bone marrow plasma capacity to induce transendothelial migration of MDA-MB231 and MCF-7 cells compared with healthy volunteers. Furthermore, patients' bone marrow mesenchymal stem cells could control the recruitment of tumour cells, modifying the MCF-7 and MDA-MB231 cell migration. In addition, we found a significantly higher MDA-MB231 cell proliferation when we used patients' bone marrow plasma compared with healthy volunteers. Interestingly, PDGF-AB, ICAM-1 and VCAM-1 levels in patients' bone marrow were significantly higher than the values of healthy volunteers, suggesting that they could be involved in the cancer cell extravasation, bone resorption and cancer cell proliferation. We believe that these results can reveal new information about what alterations happen in the bone marrow of advanced breast cancer patients before bone colonisation, changes that create optimal soil for the metastatic cascade progression.

We investigated the local immune status and its prognostic value in lung adenocarcinoma. In total, 513 lung adenocarcinoma samples from TCGA and ImmPort databases were collected and analyzed. The R package coxph was employed to mine immune-related genes that were significant prognostic indicators using both univariate and multivariate analyses. The R software package glmnet was then used for Lasso Cox regression analysis, and a prognosis prediction model was constructed for lung adenocarcinoma; clusterProfiler was selected for functional gene annotations and KEGG enrichment analysis. Finally, correlations between the RiskScore and clinical features or signaling pathways were established. Sixty-four immune-related genes remarkably correlated with patient prognosis and were further applied. Samples were hierarchically clustered into two subgroups. Accordingly, the LASSO regression algorithm was employed to screen the 14 most representative immune-related genes (PSMD11, PPIA, MIF, BMP5, DKK1, PDGFB, ANGPTL4, IL1R2, THRB, LTBR, TNFRSF1, TNFRSF17, IL20RB, and MC1R) with respect to patient prognosis. Then, the prognosis prediction model for lung adenocarcinoma patients (namely, the RiskScore equation) was constructed, and the training set samples were incorporated to evaluate the efficiency of this model to predict and classify patient prognosis. Subsequently, based on functional annotations and KEGG pathway analysis, the 14 immune-related genes were mainly enriched in pathways closely associated with lung adenocarcinoma and its immune microenvironment, such as cytokine-cytokine receptor interaction and human T-cell leukemia virus 1 infection. Furthermore, correlations between the RiskScore and clinical features of the training set samples and signaling pathways (such as p53, cell cycle, and DNA repair) were also demonstrated. Finally, the test set sample data were employed for independent testing and verifying the model. We established a prognostic prediction RiskScore model based on the expression profiles of 14 immune-related genes, which shows high prediction accuracy and stability in identifying immune features. This could provide clinical guidance for the diagnosis and prognosis of different immunophenotypes, and suggest multiple targets for precise advanced lung adenocarcinoma therapy based on subtype-specific immune molecules.

We retrospectively reviewed 100 patients who were posterior stabilized without graft fusion. Using the Sextant system, 22 patients underwent minimally invasive short-segment 4-pedicle screw fixation (<em>MIF</em>4) and 39 patients underwent minimally invasive short-segment combined with intermediate screws fixation, that is, 6-pedicle screw fixation (<em>MIF</em>6). The conventional open posterior short-segment 4-pedicle screw fixation (OPF4) technique was used in 39 patients.

To evaluate the feasibility, safety, and efficacy of percutaneous pedicle screw fixation using the Sextant system in the treatment of traumatic thoracolumbar fractures compared with the conventional open posterior short-segment pedicle screw fixation technique.

To the best of our knowledge, the clinical and radiographic outcomes of <em>MIF</em>4, <em>MIF</em>6 with polyaxial pedicle screws, and OPF4 with monoaxial pedicle screws have not been compared in the treatment of thoracolumbar fractures.

Visual analogue scores (VAS), Oswestry disability index (ODI) scores, clinical outcomes including surgical blood loss, operation time, and postoperative hospital stay, sagittal Cobb angle, vertebral body angle, and anterior height of the fractured vertebrae were compared among the 3 groups.

Significant postoperative improvements, relative to baseline, were observed in the VAS and ODI scores (P<0.05 each). There were no significant differences between the <em>MIF</em>4 and <em>MIF</em>6 groups in clinical outcomes, including surgical blood loss, operation time, postoperative hospital stay, VAS, and ODI scores (P>0.05 each). However, there were significant differences between both <em>MIF</em> groups and the OPF group (P<0.05 each). Significant improvements were observed in the sagittal Cobb angle, vertebral body angle, and anterior height of the fractured vertebrae (P<0.05 each). During follow-up, however, the correction loss of the sagittal Cobb angle was smallest in the <em>MIF</em>6 group (P<0.05).

Minimally invasive posterior stabilization using the Sextant system resulted in reduced injury compared with the open surgery, during both the internal fixation surgery and the implant removal surgery. Percutaneous screw fixation through the pedicle of the fractured vertebra is superior to the conventional OPF4 technique in correcting kyphotic deformities, and can be performed without any extra procedures.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown.

OBJECTIVE

The present study evaluated MIF in regulating platelet survival and thrombotic potential.

RESULTS

MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1α), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737-induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7(-/-) murine embryonic cells but pronounced in CXCR7-transfected Madin-Darby canine kidney cells. This prosurvival effect was attributed to the MIF-CXCR7-initiated PI3K-Akt pathway. MIF induced CXCR7-Akt-dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt(-/-) platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt(-/-) mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7.

CONCLUSIONS

MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation.

Inflammatory changes in the gastric mucosa are commonly observed in Japanese patients with functional dyspepsia (FD). However, detailed data regarding the relationship between the genetic regulatory factors of inflammation and FD are not available. We investigated the associations between FD and genetic polymorphisms of molecules associated with inflammation or immune response (IL-17A, -17F and MIF). The study was performed with 278 subjects (188 with no upper abdominal symptoms and 90 with FD according to the Roma III criteria). We employed the PCR-SSCP (multiplex PCR for IL-17A and -17F) method to detect the gene polymorphisms. Overall, the polymorphisms of the IL-17A, -17F and MIF genes were not correlated with the susceptibility to FD. However, the MIF -173C allele carrier had a significantly increased risk for the development of epigastric pain syndrome (EPS) of FD (OR, 2.12; 95% CI, 1.00-4.49; p=0.0497). In Helicobacter pylori (H. pylori)-infected cases, the number of IL-17F 7488T alleles was positively correlated with the development of EPS (OR, 11.3; 95% CI, 1.23-103.2; p=0.032), while the IL-17F T/T homozygote and the MIF -173C carrier had an increased risk for EPS (OR, 10.4; 95% CI, 1.17-92.3; p=0.036 and OR, 3.66; 95% CI, 1.19-11.3; p=0.024, respectively). In addition, a significant interaction between the IL-17F 7488 polymorphism and H. pylori infection was shown to increase the activity and inflammation scores (p=0.043 and 0.042, respectively). There were no significant associations between the IL-17A polymorphism and FD. Our results provide the first evidence that the IL-17F and MIF gene polymorphisms are significantly associated with the development of FD, particularly EPS, a subgroup of FD, in H. pylori-infected subjects. The genetic polymorphisms of inflammation or immune response-related molecules are involved in the development of one of the FD subgroups via H. pylori-induced gastric inflammation.

OBJECTIVE

A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study, we sought to determine whether antiprogestin treatment, used as a monotherapy or in combination with antiestrogen therapy, induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells.

METHODS

MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant), and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays, cell counting, flow cytometry, and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase.

RESULTS

All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity, including resistance to physiological levels of 4-OHT. Importantly, all of the clones were sensitive to the antiprogestin MIF, whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation, G(1) arrest, and apoptosis preceded by caspase activation.

CONCLUSIONS

We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+), 4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and, ultimately, improve the therapeutic index of antiestrogen therapy.

Serology is often used to diagnose acute infections by Chlamydia pneumoniae. In this study paired sera from patients with acute respiratory tract infection during an epidemic of C. pneumoniae infections were examined by five different antibody tests. These tests were the complement fixation (CF) test, the microimmunofluorescence (MIF) test, a recombinant enzyme immunoassay (rEIA) (Medac) based on a recombinant lipopolysaccharide of chlamydia and measuring antibodies to a common chlamydial antigen, and two tests that utilize preparations of C. pneumoniae organisms, the SeroCp-EIA (Savyon) (with preserved lipopolysaccharide) and the LOY-EIA (Labsystems) (without this antigen). Both of the last two tests should measure specific antibodies to C. pneumoniae, although cross-reacting antibodies may also be detected by the SeroCp-EIA. Acute infection of C. pneumoniae was serologically confirmed in 44% of the cases by at least two different tests. Using an expanded "gold standard," i.e., the presence of significant reactions in at least two tests, the sensitivity of the CF test was 69%, that of the MIF test was 88%, that of the rEIA was 89%, that of the LOY-EIA was 96%, and that of the SeroCp-EIA was 92%. Specificity was high for all methods, but adjustments of diagnostic criteria were made to several of the tests. The basis for these adjustments and supportive data are presented. Infections of C. pneumoniae were detected in patients from 8 to 83 years of age. Two peaks in the incidence of such infections were observed: one among young teenagers and a second in adults 30 to 45 years of age, corresponding to parents of young teen-agers. The tests were equally sensitive in different age groups. Reinfections seemed to be rare.

The maximal strengths of knee extensor (E) and flexor (F) muscles were compared in a group of 6 male subjects aged 24-31 years. Cross-sectional area (CSA) of E and F was evaluated from planimetric measurements of Nuclear Magnetic Resonance (NMR) imaging axial scans, carried out at five levels along the thigh. Maximal CSA for E was found at 2/3 upper femur height and at 1/3 lower femur height for F. Maximum isometric force (MIF) of E was found to be 135% greater than that of F. The maximum CSA of E was found to be 93% larger than CSA of F. The calculated mechanical advantage of the flexors was estimated to be 13.8% higher than that of the knee extensors (0.116 +/- 0.012 and 0.132 +/- 0.005, respectively). However, when MIF of E and F were standardised for their respective CSA, no significant difference was found between their stress: 80.1 +/- 15.5 N.cm-2 for E and 70.5 +/- 7.0 N.cm-2 for F. From the present study, it is concluded that no significant difference exists between the maximum stress of knee extensor and flexor muscles despite large differences in their absolute values of force and CSA and that the NMR imaging technique enables accurate in-vivo determination of the CSA of individual muscles.

Exposure of isolated Xenopus animal pole ectoderm to the XTC mesoderm-inducing factor (XTC-MIF) causes the tissue to undergo gastrulation-like movements. In this paper, we take advantage of this observation to investigate the control of various aspects of gastrulation in Xenopus. Blastomeres derived from induced animal pole regions are able, like marginal zone cells, but unlike control animal pole blastomeres, to spread and migrate on a fibronectin-coated surface. Dispersed animal pole cells are also able to respond to XTC-MIF in this way; this is one of the few mesoderm-specific responses to induction that has been observed in single cells. The ability of induced animal pole cells to spread on fibronectin is abolished by the peptide GRGDSP. However, the elongation of intact explants is unaffected by this peptide. This may indicate that fibronectin-mediated cell migration is not required for convergent extension. We have investigated the molecular basis of XTC-MIF-induced gastrulation-like movements by measuring rates of synthesis of fibronectin and of the integrin beta 1 chain in induced and control explants. No significant differences were observed, and this suggests that gastrulation is not initiated simply by control of synthesis of these molecules. In future work, we intend to investigate synthesis of other integrin subunits and to examine possible post-translational modifications to fibronectin and the integrins.

Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI.

Three commercially available, peptide-based enzyme-linked immunosorbent assay (ELISA) systems (Chlamydia trachomatis IgG and IgA EIA [CT-EIA; Labsystems OY, Helsinki, Finland], SeroCT IgG and IgA [SeroCT; Savyon Diagnostics Ltd., Ashdod, Israel], and Chlamydia trachomatis IgG and IgA pELISA [CT pELISA; Medac, Wedel, Germany]) were evaluated for the detection of serum immunoglobulin G (IgG) and IgA antibodies specific for Chlamydia trachomatis and compared to the "gold standard" assay, the microimmunofluorescence (MIF) assay. Serological responses were analyzed in 149 women aged 20 to 30 years. Cervical swabs obtained from these women were examined for C. trachomatis by PCR, and 43 were found to be positive. The overall seroprevalence rates detected by CT-EIA, SeroCT, CT pELISA, and the MIF assay were 42, 42, 35, and 39%, respectively, for IgG and 7, 7, 3, and 7%, respectively, for IgA. The IgG seroprevalence rates for the PCR-positive women were two to three times higher than those for the PCR-negative women, i.e., 72 versus 29%, 72 versus 29%, 47 versus 26%, and 74 versus 25% for CT-EIA, SeroCT, CT pELISA, and the MIF assay, respectively. After discrepancy analysis, the sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the IgG assays; for CT-EIA they were 84.7, 98.6, 98.4, and 86.7%, respectively; for CT pELISA they were 71.4, 97.3, 96.2, and 78.3%, respectively; for SeroCT they were 84.7, 98.6, 98.4, and 86.3%, respectively; and for the MIF assay they were 79.2, 83.1, 98.3, and 83.1%, respectively. In conclusion, these peptide-based ELISA systems for the serological detection of C. trachomatis infection performed as well as the MIF assay. Since these tests are less time-consuming, less expensive, and easier to perform than the MIF assay, they might be useful in the serodiagnosis of chlamydial infection.