Leukocyte Inhibitory Factor Research Articles

#1089 Urinary Protein Biomarkers for Assessing Disease Activity and Chronicity in IgA Nephropathy

Abstract Background and Objectives IgA nephropathy (IgAN) is a progressive kidney disease characterized by the mesangial deposition of galactose-deficient IgA1, leading to kidney damage and may eventually lead to end-stage kidney disease. Current treatment decisions for IgAN patients rely on the degree of proteinuria. However, this is a non-specific parameter that can be the result of inflammatory disease activity and/or chronic glomerular damage. In this study, we employed the Olink proximity extension assay to comprehensively analyze the urinary proteome of IgAN patients. Our goal was to identify biomarkers capable of distinguishing between IgAN disease activity and disease chronicity. Method Urine samples were collected from 41 IgAN patients and 41 age- and eGFR-matched controls. Patient characteristics are shown in Table 1. Using Olink Proteomics, we measured 276 proteins in urine, from the inflammation, organ damage, and cardiometabolic panels. Comparisons were made between IgAN patients and controls, as well as between those with mild (eGFR ≥ 60 ml/min/1.73 m2 and uPCR < 100 mg/mmol) and active IgAN (eGFR ≥ 60 ml/min/1.73 m2 and uPCR ≥ 100 mg/mmol) to identify biomarkers for disease activity. Additionally, we compared IgAN patients with eGFR ≥ 60 ml/min/1.73 m2 to those with eGFR ≤ 30 ml/min/1.73 m2 to identify biomarkers associated with disease chronicity. Mann-Whitney U tests were used for group comparisons. Correction for multiple testing was performed using the Benjamini-Hochberg procedure. Disease activity and chronicity scores were calculated by averaging the z-scores of the identified biomarkers. Results L-Selectin (SELL), leukocyte inhibitory factor (LIF), interleukin-6 (IL-6), and carbonic anhydrase 1 (CA1) were most prominently elevated in IgAN patients compared to controls (Fig. 1A). SELL and CA1 were also markedly elevated in the active versus mild IgAN patients, and were therefore selected as biomarkers of disease activity (Fig. 1B). Contactin 2 (CNTN2) and chemokine (C-C motif) ligand 25 (CCL25) were most prominently elevated in IgAN patients with eGFR ≤ 30 ml/min/1.73 m2, and were selected as biomarkers of disease chronicity (Fig. 1C). We plotted the disease activity and chronicity scores calculated from the identified biomarkers to distinguish the different IgAN disease stages (Fig. 1D). Conclusion This study identified urine biomarkers indicative of IgAN disease activity and chronicity. Calculating an activity and chronicity score using these biomarkers offers a new approach to distinguish between inflammatory disease activity and chronicity, providing an alternative to proteinuria for guiding treatment decisions in IgAN patients. Validation of these biomarkers, and the scores calculated from them, in a larger cohort of IgAN patients is currently underway.

A specific combination of laboratory data is associated with overweight lungs in patients with COVID-19 pneumonia at hospital admission: secondary cross-sectional analysis of a randomized clinical trial.

Lung weight may be measured with quantitative chest computed tomography (CT) in patients with COVID-19 to characterize the severity of pulmonary edema and assess prognosis. However, this quantitative analysis is often not accessible, which led to the hypothesis that specific laboratory data may help identify overweight lungs. This cross-sectional study was a secondary analysis of data from SARITA2, a randomized clinical trial comparing nitazoxanide and placebo in patients with COVID-19 pneumonia. Adult patients (≥18 years) requiring supplemental oxygen due to COVID-19 pneumonia were enrolled between April 20 and October 15, 2020, in 19 hospitals in Brazil. The weight of the lungs as well as laboratory data [hemoglobin, leukocytes, neutrophils, lymphocytes, C-reactive protein, D-dimer, lactate dehydrogenase (LDH), and ferritin] and 47 additional specific blood biomarkers were assessed. Ninety-three patients were included in the study: 46 patients presented with underweight lungs (defined by ≤0% of excess lung weight) and 47 patients presented with overweight lungs (>0% of excess lung weight). Leukocytes, neutrophils, D-dimer, and LDH were higher in patients with overweight lungs. Among the 47 blood biomarkers investigated, interferon alpha 2 protein was higher and leukocyte inhibitory factor was lower in patients with overweight lungs. According to CombiROC analysis, the combinations of D-dimer/LDH/leukocytes, D-dimer/LDH/neutrophils, and D-dimer/LDH/leukocytes/neutrophils achieved the highest area under the curve with the best accuracy to detect overweight lungs. The combinations of these specific laboratory data: D-dimer/LDH/leukocytes or D-dimer/LDH/neutrophils or D-dimer/LDH/leukocytes/neutrophils were the best predictors of overweight lungs in patients with COVID-19 pneumonia at hospital admission. Brazilian Registry of Clinical Trials (REBEC) number RBR-88bs9x and ClinicalTrials.gov number NCT04561219.

Fractalkine Improves the Expression of Endometrium Receptivity-Related Genes and Proteins at Desferrioxamine-Induced Iron Deficiency in HEC-1A Cells.

Fractalkine (CX3CL1/FKN) is a unique chemokine belonging to the CX3C chemokine subclass. FKN exists in two forms: a membrane-bound form expressed by both endometrium cells and trophoblasts thought to be implicated in maternal-fetal interaction and a soluble form expressed by endometrium cells. Endometrium receptivity is crucial in embryo implantation and a complex process regulated by large numbers of proteins, e.g., cytokines, progesterone receptor (PR), SOX-17, prostaglandin receptors (PTGER2), and tissue inhibitors of metalloproteinases (TIMPs). It has also been reported that iron is important in fertility and affects the iron status of the mother. Therefore, iron availability in the embryo contributes to fertilization and pregnancy. In this study, we focused on the effect of iron deficiency on the secreted cytokines (IL-6, IL-1β, leukocyte inhibitory factor, TGF-β), chemokines (IL-8, FKN), and other regulatory proteins (bone morphogenic protein 2, activin, follistatin, PR, SOX-17, prostaglandin E2 receptor, TIMP2), and the modifying effect of FKN on the expression of these proteins, which may improve endometrium receptivity. Endometrial iron deficiency was mediated by desferrioxamine (DFO) treatment of HEC-1A cells. FKN was added to the cells 24 h and 48 h after DFO with or without serum for modelling the possible iron dependence of the alterations. Our findings support the hypothesis that FKN ameliorates the effects of anemia on the receptivity-related genes and proteins in HEC-1A cells by increasing the secretion of the receptivity-related cytokines via the fractalkine receptor (CX3CR1). FKN may contribute to cell proliferation and differentiation by regulating activin, follistatin, and BMP2 expressions, and to implantation by altering the protein levels of PR, SOX-17, PTGER2, and TIMP2. FKN mitigates the negative effect of iron deficiency on the receptivity-related genes and proteins of HEC-1A endometrium cells, suggesting its important role in the regulation of endometrium receptivity.

Autocross-linked hyaluronic acid gel and adipose-derived mesenchymal stem cell composites for the treatment intrauterine adhesions

ObjectiveTo evaluate the effect of adding adipose-derived mesenchymal stem cells (ASCs) to autocross-linked hyaluronic acid (HA) gel for intrauterine adhesion (IUA) treatment. MethodsA rat IUA model was established by mechanical curettage and infection, and then different treatments were administered to the rats on day 7 after modeling. Ninety-six rats were randomly divided into the following groups: IUA model group, gel therapy group, and combination therapy group (HA gel combined with ASCs). Eight rats per group were sacrificed on days 7, 10, 14 and 21 for the subsequent experiments. Morphological changes were determined by hematoxylin-eosin staining and Masson staining. Smad3 and leukocyte inhibitory factor (LIF) were analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. ResultsThe endometrial lines in the gel therapy group and the combination therapy group were more complete than those in the model group. Masson staining showed that fibrosis area rates in the gel therapy group and the combination therapy group were significantly lower than those in the model group on day 7(P < 0.05). During the observation period, the fibrosis area rates in the combination therapy group remained lower than those in the model group (P < 0.05). The mRNA expression of Smad3 in the combination therapy group was lower than that in the model group and gel therapy group during the observation period (P < 0.05). The mRNA expression level of LIF in the combination therapy group was higher than that in the model group and the gel therapy group throughout the observation period (P < 0.05). ConclusionsHA gel was effective in preventing the IUA adhesion formation at the early stage of the observation period, while ASC enhanced this effect throughout the observation period. Gel and ASC composites helped to improve endometrial receptivity.

Tumor necrosis factor-alpha aggravates gliosis and inflammation of activated retinal Müller cells

Tumor necrosis factor-alpha (TNF-α), a major inflammatory factor released from activated retinal glial cells, is implicated in the pathogenesis of glaucoma. In this study, we investigated whether and how TNF-α may affect functional conditions of activated retinal Müller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Müller cells, TNF-α treatment aggravated cell gliosis, as evidenced by significantly increased expression of glial fibrillary acidic protein (GFAP). TNF-α treatment of the DHPG-activated Müller cells decreased cell proliferation and induced cell apoptosis. In normal Müller cells, TNF-α treatment increased the mRNA levels of leukocyte inhibitory factor (LIF), intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and chemokine C–C-motif ligand 2 (CCL2), which could be significantly attenuated when Müller cells were pre-activated. However, TNF-α-induced elevation in mRNA levels of inflammatory factors, such as TNF-α, inducible nitric oxide synthase (iNOS), and interleukin-6 (IL-6), in normal Müller cells still kept higher levels when Müller cells were pre-activated. Furthermore, the TNF-α-induced changes of cytokines were partially mediated by NF-κB signaling pathway. Our results suggest that TNF-α may promote gliosis and inflammatory response of activated Müller cells, thus aggravating RGC injury in glaucoma.

RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition

Objective To observe RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment. Methods Retinal vascular endothelial cells were cultured in vitro, and the logarithmic growth phase cells were used for experiments. The cells were divided into the control group and high glucose group. The cells of two groups were cultured for 5 hours with 5, 25 mmol/L glucose, respectively. And then, whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq. Now with biological big data obtained as a basis, to analyze the differentially expressed genes (DEGs). And through enrichment analysis to explain the differential functions of DEGs and their signal pathways. Results The gene expression profiles of the two groups of cells were obtained. Through analysis, 449 DEGs were found, including 297 upregulated and 152 downregulated ones. The functions of DEGs were influenced by regulations over molecular biological process, cellular energy metabolism and protein synthesis, etc. Among these genes, ITGB1BP2, NCF1 and UNC5C were related to production of inflammation; AKR1C4, ATP1A3, CHST5, LCTL were related to energy metabolism of cells; DAB1 and PRSS55 were related to protein synthesis; SMAD9 and BMP4 were related to the metabolism of extracellular matrix. GO enrichment analysis showed that DEGs mainly act in three ways: regulating biological behavior, organizing cellular component and performing molecular function, which were mainly concentrated in the system generation of biological process part and regulation of multicellular organisms. Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in transforming growth factor-β (TGF-β) signaling pathway, complement pathway and amino acid metabolism-related pathways have also been affected, such as tryptophan, serine and cyanide. Among them, leukocyte inhibitory factor 9 and bone morphogenetic protein 4 play a role through the TGF-β signaling pathway. Conclusions High glucose affects the function of retinal vascular endothelial cells by destroying transmembrane conduction of retinal vascular endothelial cells, metabolism of extracellular matrix, and transcription and translation of proteins. Key words: Retinal Vessels/cytology; Endothelial cells; Gene expression profiling; Diabetic retinopathy/etiology

Literature Review on the Role of Uterine Fibroids in Endometrial Function.

Uterine fibroids are benign uterine smooth muscle tumors that are present in up to 8 out of 10 women by the age of 50. Many of these women experience symptoms such as heavy and irregular menstrual bleeding, early pregnancy loss, and infertility. Traditionally believed to be inert masses, fibroids are now known to influence endometrial function at the molecular level. We present a comprehensive review of published studies on the effect of uterine fibroids on endometrial function. Our goal was to explore the current knowledge about how uterine fibroids interact with the endometrium and how these interactions influence clinical symptoms. Our review shows that submucosal fibroids produce a blunted decidualization response with decreased release of cytokines critical for implantation such as leukocyte inhibitory factor and cell adhesion molecules. Furthermore, fibroids alter the expression of genes relevant for implantation, such as bone morphogenetic protein receptor type II, glycodelin, among others. With regard to heavy menstrual bleeding, fibroids significantly alter the production of vasoconstrictors in the endometrium, leading to increased menstrual blood loss. Fibroids also increase the production of angiogenic factors such as basic fibroblast growth factor and reduce the production of coagulation factors resulting in heavy menses. Understanding the crosstalk between uterine fibroids and the endometrium will provide key insights into implantation and menstrual biology and drive the development of new and innovative therapeutic options for the management of symptoms in women with uterine fibroids.

Effect of leukocyte inhibitory factor on neuron differentiation from human induced pluripotent stem cell-derived neural precursor cells.

Direct derivation of human induced pluripotent stem cells into neural precursor cells and differentiation of these into neurons holds great promise in the cell therapy of neuro-degenerative diseases. However, the availability and survival rate of neurons requires improvement. In the present study, it was found that the addition of 5 ng/ml leukocyte inhibitory factor (LIF) during the process of differentiation significantly improved the expression of neuron-specific class III β-tubulin (TUJ1) and microtubule-associated protein 2 (MAP2), as detected by immunofluorescence and western blotting. In addition, LIF improved the cell viability, increased the expression of phosphorylated-protein kinase B (AKT), downregulated the expression of proinflammatory cytokines, including interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), and upregulated the expression of anti-inflammatory cytokines, including interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). After adding the phosphatidylinositol 3-kinase (PI3K)/AKT signaling inhibitor LY294002 or wortmannin to the LIF differentiation group, LIF-induced changes in the protein expression of TUJ1 and MAP2 were reversed, but this effect could not be prevented by rapamycin, a mechanistic target of rapamycin signaling inhibitor. The expression of cytokines associated with inflammation and cell viability was reversed by LY294002 and wortmannin, but not by rapamycin. In conclusion, LIF could improve neuronal differentiation and survival through the activation of PI3K/AKT signaling and the anti-inflammatory effect. The anti-inflammatory effect may be mediated by the activation of PI3K/AKT.

Stem Cell Biology and its Role in Regenerative Medicine: A Concept Shaping the Future of Medicine

Stem cells are special type of cells that have unique capacity to renew themselves and differentiate to give rise to specialized cell types for many tissues. Hence, they are the principal cells of all cell types. Stem cells can be embryonic, fetal, and adult or somatic types. Cells can be reprogrammed artificially through the introduction of endogenous transcription factors by using viruses like retrovirus to produce iPSCs (induced pluripotent stem cells). There are proteins that are important in the regulation of stem cell differentiation such as bone morphogenic protein -2 (BMP-2), leukocyte inhibitory factor and micro RNAs. Cutting edge research featuring stem cells will gear us to medical discovery of cell-based therapies of various diseases occurring in animals and human beings. Apart from applications of stem cells there is a widespread controversy over the use of embryonic stem cells. The controversy targets the techniques used to derive new embryonic stem cell lines which damage the blastocyst. However, reprogramming of adult cells into iPSCs holds great promise for regenerative medicine. Advancement in stem cell research is ever growing and now a day it is gaining a concern and this is a promising indication that adult and embryonic stem cells will be used in the future to treat cancer, paralysis, cardiac failure, stroke, diabetes and many other diseases. In this paper we reviewed the properties and classification of stem cells, regulatory factors of stem cell pluripotency and differentiation, reprograming and potential clinical applications of stem cells, as well as ethical issues in stem cell therapy.

Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand?

Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options.

Localization of the immune response in sarcoidosis.

Pulmonary sarcoidosis is an interstitial disease characterized by granulomas within the lung parenchyma, anergy to a variety of skin tests, and decreased numbers of circulating T-lymphocytes. To evaluate the effector cell populations present at sites of disease in patients with active pulmonary sarcoidosis, inflammatory and immune effector cells were isolated from lung via bronchoalveolar lavage and compared to comparable cell populations from the peripheral blood of the same patients and similar cell populations of normal subjects and patients with idiopathic pulmonary fibrosis. Patients with sarcoidosis had a marked increase in the percentage of T-lymphocytes in the lung despite a significant peripheral blood T-lymphocytopenia. In addition, many of these T-lymphocytes demonstrated surface marker characteristics associated with lymphocyte activation, and they spontaneously secreted leukocyte inhibitory factor. In contrast to patients with sarcoidosis, normal subjects and patients with idiopathic pulmonary fibrosis had similar percentages of T-lymphocytes in lung and blood, and there was no evidence for T-lymphocyte activation. Analysis of lymphocytes in uninvolved marrow from 7 of 8 patients with sarcoidosis revealed proportions of T-lymphocytes similar to those in the marrows of normal subjects and patients with idiopathic pulmonary fibrosis. In comparison, one patient with sarcoidosis had large numbers of T-lymphocytes in bone marrow, but only in areas where there were granulomas in the marrow. These studies suggest that: (1) the alveolitis of pulmonary sarcoidosis is characterized by large numbers of activated T-lymphocytes, and (2) there is an anatomic localization of the immune response in sarcoidosis in that analysis of uninvolved tissues such as peripheral blood may not reflect local immune responses at sites of granuloma formation.

Gene expression profiling of human mesenchymal stem cells chemotactically induced with CXCL12

In situ tissue engineering is a promising approach in regenerative medicine, with the possibility that adult stem or progenitor cells will be guided chemotactically to a tissue defect and subsequently differentiate into the surrounding tissue type. Mesenchymal stem cells (MSC) represent attractive candidate cells. Chemokines such as CXCL12 (SDF-1alpha) chemoattract MSC, but little is known about the molecular processes involved in the chemotaxis and migration of MSC. In this study, MSC recruitment by CXCL12 was investigated by genome-wide microarray analysis. The dose-dependent migration potential of bone-marrow-derived MSC toward CXCL12 was measured in an in vitro assay, with a maximum being recorded at a concentration of 1,000 nM CXCL12. Microarray analysis of MSC stimulated with CXCL12 and non-stimulated controls showed 30 differentially expressed genes (24 induced and six repressed). Pathway analysis revealed 11 differentially expressed genes involved in cellular movement and cytokine-cytokine receptor interaction, including those for migratory inducers such as the chemokines CXCL8 and CCL26, the leukocyte inhibitory factor, secretogranin II, and prostaglandin endoperoxide synthase 2. These results were confirmed by real-time polymerase chain reaction for selected genes. The obtained data provide further insights into the molecular mechanisms involved in chemotactic processes in cell migration and designate CXCL12 as a promising candidate for in situ recruitment in regenerative therapies.

The G->c polymorphism of the p53 gene is strongly associated with decreased pregnancy rates in fresh IVF patients via an ovarian mechanism

OBJECTIVE: p53 is a key tumor suppressor gene responsible for the repair of damaged DNA. A polymorphism of the p53 gene at codon 72 (Arg→Pro) decreases p53 activity and may lead to an increase in DNA damage during oogenesis. Protein products of the p53 gene have also been shown to promote leukocyte inhibitory factor (LIF) production in the murine model and its reduction in p53 null mice significantly decreases litter size. We hypothesize that women who carry the p53c72 (pro/pro) polymorphism of the p53 gene may experience decreased implantation rates, either through diminished LIF production at the level of the endometrium or through diminished oocyte quality from inadequate DNA repair mechanisms. DESIGN: Prospective analysis of oocyte donor recipients and young women (<35) with unexplained infertility. Incidence of the p53c72 Pro (variant) polymorphism compared to p53c72 Arg (wild type) was assessed as well as IVF outcome. MATERIALS AND METHODS: A total of one hundred and sixty three samples were collected from donor egg recipients (DER) (n=76) and patients <35 years of age (n=87) with unexplained infertility. DNA was extracted and polymerase chain reaction amplification of p53c72 was performed. The frequency of each variant was compared to known population controls. Oocyte yield, implantation rates, and pregnancy rates were analyzed. RESULTS: The prevalence of the variant polymorphism was increased in our infertile population compared to the general population (47% vs. 35%, p<0.05). In the DER group, there was no difference in average age, implantation rates (52% vs. 48%) or pregnancy rates (78% vs. 85%) for patients with the variant polymorphism compared to the wild type, respectively. In the fresh IVF group, there was no difference baseline characteristics, however, within this group those with the polymorphism indicating lower p53 activity had decreased oocyte yield (9.31± 4.1 vs.12.8±5.3, p<0.05), decreased implantation rates (16% vs. 44%, p<0.01), and decreased pregnancy rates 30% vs.68% p<0.01) compared to the wild type. CONCLUSIONS: We have found that women who are homozygous for the p53c72Pro variant have both a decreased ovarian response to exogenous gonadotropins and decreased pregnancy rates when compared to the wild type. This suggests that p53 activity is crucial to oogenesis and early embryogenesis. The lack of impact of p53 activity on the DER population suggests that other cytokines may help overcome any negative impact of this polymorphism at the level of the endometrium.

Rapid and Irreversible Activation of MAPK Kinase Pathway during Myelopoiesis.

Mononuclear phagocytes are essential for innate immunity as well as wound healing, tissue remodeling and angiogenesis. We hypothesize that the state of macrophage maturation may prolong chronic inflammation. Despite finding that normal macrophage maturation is disrupted in some myeloid leukemias and the identification of several signal transduction pathways involved in myelopoiesis, little is known about steps involved in this maturational pathway. We examined the pathways responsible for macrophage differentiation in the myeloid leukemic THP-1 cell line either treated with PMA (to induce differentiation) or with DMSO (vehicle) as a model. We found that treatment of THP-1 cells with PMA resulted in cells becoming macrophage-like as indicated by cellular adherence and expression of cell surface antigens. To correspond with surface expression of CD68, MHC II, CD11b, mannose receptor and CD14, we further characterized macrophage differentiation at the molecular level. Based on microarray and proteomics data comparing human monocytes to macrophages, we selected several genes to monitor during the process of myeloid differentiation. By RT-PCR analysis, we found increase expression of cathespins B and H, metalloproteinases (MMP9 and MMP2), and interleukin 4 inducible gene 1 (IL4-i1), whereas expression of leukocyte inhibitory factor (LEI) and calgranulin B decreased during differentiation. Furthermore, PMA-induced differentiation of THP-1 cells required only 15 min stimulation. We next examined the intracellular pathways mediating differentiation of THP1 cells. In the presence of PMA and the Mek inhibitor, U0126, THP-1 cells failed to become adherent, did not express macrophage surface antigens, did not upregulate expression of MMP9 or IL4-il, and calgranulin B expression was maintained indicating that the cells were still immature. However, when cells were pretreated with PMA for 1 hr prior to adding U0126, the cells continued to differentiate. Cytosolic and nuclear fractions were isolated from cells treated with PMA in the absence or presence of U0126 and found that U0126 did not prevent the phosphorylation of Erk1/2 but inhibited their nuclear translocation. In the absence of pErk1/2 nuclear translocation in the U0126-treated cells, there was no increase in expression of p21CIP1, indicating that cell arrest did not occur. Since maturation of these cells was associated with the nuclear translocation of both Erk1/2, we were interested in determining which Erk isoform was essential for differentiation. THP-1 cells were transiently transfected with either DN-Erk1 or DN-Erk2 or both then treated with PMA. There was no change in cellular adherence when DN-Erk1 was expressed alone but there was a two-fold decrease in adherence in the DN-Erk2 transfected cells as measured by crystal violet staining. Since MAPK kinase pathway was critical in regulating THP-1 differentiation, we examined whether this was conserved in GM-CSF-induced human peripheral blood monocyte (PBM) maturation to macrophages. We found that GM-CSF-induced the phosphorylation of Erk1/2 within 15 min of treatment and was still detectable at d3 and d7. Treatment of PBM with U0126 inhibited GM-CSF-induced differentiation. Similar to that seen in THP-1 cells, U0126 failed to inhibit differentiation in PBM pretreated with GM-CSF for 1h. Our observations suggest that commitment to differentiate occurs very rapidly and is irreversible under certain conditions. Dissection of the role of Mek and Erk kinases in myeloid differentiation may lead to the identification of therapeutic targets in myeloid leukemia.

Cdc42 plays a critical role in assembly of sarcomere units in series of cardiac myocytes

Cardiomyocyte hypertrophy is observed in various cardiovascular diseases and causes heart failure. We here examined the role of small GTP-binding proteins of Rho family in phenylephrine (PE)-or leukocyte inhibitory factor (LIF)-induced hypertrophic morphogenesis of cultured neonatal rat cardiomyocytes. Both LIF and PE increased cell size of cardiomyocytes. LIF induced an increase in the length/width ratio of cardiomyocytes, while PE did not change the ratio. Adenoviral gene transfer of constitutively active mutants of Cdc42 increased the length/width ratio of cardiomyocytes and dominant negative mutants of Cdc42 conversely inhibited LIF-induced cell-elongation, while mutants of RhoA and Rac1 did not affect the length/width ratio of cardiomyocytes. These results suggest that Cdc42, but not RhoA and Rac1, is involved in LIF-induced sarcomere assembly in series in cardiomyocytes.

Enhancement of immunogenicity of JEG‐3 Cells by ectopic expression of HLA‐A*0201 and CD80

The chorioncarcinoma cell line JEG‐3 escapes immunity by secretion of leukocyte inhibitory factor suppressing leukocyte proliferation. The cells lack expression of classical HLA alleles but express nonclassical HLA‐G, which binds to killer inhibitory receptor of natural killer cells, preventing cytolysis. We investigated whether JEG‐3 cells are capable of immune stimulation after introduction of classical HLA and T‐cell costimulatory signals. JEG‐3 cells were transduced with vectors for HLA‐A*0201 and/or CD80. Parental JEG‐3, or JEG‐3/A2, JEG‐3/CD80, or JEG‐3/A2/CD80 were used to stimulate allogeneic T cells. While parental JEG‐3 cells induced only marginal proliferation of resting T cells, HLA‐A2 or CD80 expressing JEG‐3 induced enhanced proliferation. Double transfectants were most efficient. This difference was more obvious when T cells were preactivated by PHA. T cell lines restimulated with JEG‐3 transfectants were characterized for expansion, phenotypes, and cytolytic activity. HLA‐A2 matched and nonmatched donors were compared. T cells stimulated with JEG‐3/A2 or JEG‐3/CD80 were cytolytic towards parental JEG‐3 cells. Again double positive JEG‐3/A2/CD80 induced highest cytolytic activity, most obvious in HLA‐nonmatched donors suggesting alloreactivity to HLA. Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by JEG‐3 cells when classical HLA and/or costimulatory signals are present on the cells.

THE EFFECTS OF INFECTION OF THERMAL INJURY BY PSEUDOMONAS AERUGINOSA PAO1 ON THE MURINE CYTOKINE RESPONSE

Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death. Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P. aeruginosa PAO1. Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1β, macrophage inflammatory protein (MIP)-1β and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) expression. The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines [stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-α]; induced the expression of granulocyte–macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5h and the expression of additional cytokines, including TGF-β, TNF-β, lymphotoxin β (LT-β), interferon γ (IFN-γ), and IFN-β by 40h post-burn/infection. While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40h post-burn/infection. These results suggest that in P. aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P. aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism.

Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative.

We previously reported that combined treatment with bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-4 (FGF-4) induces cardiogenic events culminating in full cardiac differentiation of non-precardiac mesoderm explanted from stage 6 avian embryos (Lough et al. [1996] Dev. Biol. 178:198-202.). To elucidate the respective functions of BMP and FGF in initiating and maintaining the cardiogenic process, we have used these ectopic cells as a cardiac specification model to ascertain requirements for growth factor specificity and extent of application, as well as induction of cardiac transcription factors. The inability of some BMP isoforms to replace the inductive activity of BMPs-2/4 indicated a specific requirement for this signaling pathway; moreover, neither activin-A nor insulin, which support terminal differentiation of precardiac mesoderm, nor leukocyte inhibitory factor (LIF), which promotes hypertrophy in cardiac myocytes, could replace BMP's cardiogenic activity. A similarly specific requirement for FGF-2/4 signaling was revealed since neither FGF-7, activin-A nor insulin could replace this activity. The effect of both factors was concentration-dependent; maximal incidence of explant differentiation for each occurred at 50 ng/ml. Surprisingly, the majority of explants treated with high BMP levels (250 ng/ml) exhibited a non-cardiac phenotype that was characterized by intense expression of alkaline phosphatase, suggesting differentiation toward an alternative mesodermal phenotype. Experiments to assess the duration of exposure to each factor that was required revealed that while exposure to BMP and FGF during only the initial 30 min of a 48-hr culture period was sufficient to induce cardiogenesis in a significant percentage of explants, 100% incidence of explant differentiation was obtained only when FGF treatment was restricted to the first 30 min and BMP was continuously present during the 48-hr culture period. Treatment with both growth factors was required to induce the cardiac transcription factors cNkx-2.5 and SRF; neither mRNA was induced by BMP or FGF alone. These findings indicate that: (1) specific members of the BMP and FGF families are required to induce cardiogenesis in non-precardiac mesoderm; (2) BMPs-2/4 may function as a morphogen; (3) brief application of both factors can induce cardiogenesis in a modest number of explants whereas (4) 100% incidence of explant differentiation can only be attained by brief FGF treatment combined with continuous BMP treatment and (5) both factors are necessary to induce downstream cardiac transcription factors. These findings are interpreted in terms of these factors' possible roles during cardiac specification and differentiation.

Different pattern of cytokine production and mRNA expression by lymphoid and non-lymphoid cells isolated from human palatine tonsil.

To investigate the cytokines involved in the interaction between circulating (B and T lymphocytes) and non-circulating (stromal cells) elements present in lymphoid tissue, highly purified populations were isolated from human tonsils and the cytokine production and mRNA expression (interleukin-1 alpha, -2, -4, -5, -6, -8, -10, leukocyte inhibitory factor, granulocyte-macrophage colony-stimulating factor, and interferon-gamma) were assessed both by immunoassay and reverse transcriptase polymerase chain reaction under resting conditions and after activation with tumor necrosis factor-alpha. Under basal conditions most cytokines were not detected, except for interleukin-8 which was produced by T lymphocytes and lymphoid cells. Activation by tumor necrosis factor-alpha induced interleukin-8 production by B lymphocytes. Tonsillar T lymphocytes expressed mRNA for interleukin-1 alpha, -8, -10, -4, leukocyte inhibitory factor, and interferon-gamma, only interleukin-4 was expressed by resting peripheral blood T lymphocytes. Tonsillar B lymphocytes were mRNA positive for interleukin-1 alpha, -8, -10, leukocyte inhibitory factor, and interferon-gamma, these were not expressed by peripheral blood B lymphocytes. Stromal cells constitutively produce interleukin-6 whose levels increased 5 times upon tumor necrosis factor-alpha activation Granulocyte-macrophage colony-stimulating factor and interleukin-8 were detected only after tumor necrosis factor-alpha activation. Only stromal cells constitutively express interleukin-6 and granulocyte-macrophage colony-stimulating factor and show a cytokine pattern different from that described for other non-lymphoid cells, such as follicular dendritic cells. These data indicate that in the human tonsil population, lymphoid and non-lymphoid cells can be distinguished by different patterns of cytokine expression.

Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism.

Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm, inhibits CD11b/CD18-dependent neutrophil adhesion by binding to CD1lb. We studied the effects of NIF on neutrophil-dependent endothelial cell injury using bovine pulmonary microvessel endothelial cells grown on microporous filters. Endothelial injury was determined as an increase in the transendothelial 125I-albumin clearance rate (a measure of transendothelial permeability). Layering of neutrophils on the endothelial cell monolayer (ratio of 10 neutrophils: 1 endothelial cell) followed by activation of neutrophils with 500 nM of phorbol 12-myristate 13-acetate (PMA) increased transendothelial permeability of albumin by 3- to 4-fold over control monolayers. Pretreatment of neutrophils with NIF at concentrations of 100 nM and above prevented the increased permeability. Pretreatment of neutrophils with the anti-CD18 monoclonal antibody (mAb) IB4 similarly prevented the increase of permeability. Pretreatment of neutrophils with OKM-1, a control isotype-matched mAb directed against an irrelevant epitope on CD11b mAb, did not affect the neutrophil-dependent increase in permeability. NIF reduced the adhesion of neutrophils at concentrations of > or = 100 nM and this effect was abolished by an anti-NIF polyclonal Ab. However, NIF did not prevent the generation of superoxide anions following PMA-induced activation of neutrophils layered on endothelial cell. These findings indicate that NIF inhibits the neutrophil-dependent endothelial injury by preventing CD11b/CD18-mediated neutrophil adhesion, but without altering the oxidant generating capacity of neutrophils interacting with the endothelial cell monolayer.