Relationship of macrophage migration inhibitory factor levels in PBMCs, lesional skin and serum with disease severity and activity in vitiligo vulgaris

Melanocyte loss in vitiligo vulgaris is believed to be an autoimmune process. Macrophage migration inhibitory factor (MIF) is involved in many autoimmune skin diseases. We determined the possible role of MIF in the pathogenesis of vitiligo vulgaris, and describe the relationship between MIF expressions and disease severity and activity. Serum MIF concentrations and mRNA levels in PBMCs were measured in 44 vitiligo vulgaris patients and 32 normal controls, using ELISA and real-time RT-PCR. Skin biopsies from 15 patients and 6 controls were analyzed by real-time RT-PCR. Values are reported as median (25th-75th percentile). Serum MIF concentrations were significantly increased in patients [35.81 (10.98-43.66) ng/mL] compared to controls [7.69 (6.01-9.03) ng/mL]. MIF mRNA levels were significantly higher in PBMCs from patients [7.17 (3.59-8.87)] than controls [1.67 (1.23-2.42)]. There was also a significant difference in MIF mRNA levels in PBMCs between progressive and stable patients [7.86 (5.85-9.13)vs 4.33 (2.23-8.39)] and in serum MIF concentrations [40.47 (27.71-46.79) vs 26.80 (10.55-36.07) ng/mL]. In addition, the vitiligo area severity index scores of patients correlated positively with changes of both serum MIF concentrations (r = 0.488) and MIF mRNA levels in PBMCs (r = 0.426). MIF mRNA levels were significantly higher in lesional than in normal skin [2.43 (2.13-7.59)vs 1.18 (0.94-1.83)] and in patients in the progressive stage than in the stable stage [7.52 (2.43-8.84)vs 2.13 (1.98-2.64)]. These correlations suggest that MIF participates in the pathogenesis of vitiligo vulgaris and may be useful as an index of disease severity and activity.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Blastocyst-endometrium interaction: intertwining a cytokine network

The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipotent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo. Studies in humans and in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproductive tract; however, the interactions between these factors that converge for successful implantation are not well understood. This review focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differentiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Serum factors inhibitory for in vitro development of Plasmodium falciparum blood-stage parasites

Sera from 29 individuals residing in a malaria-endemic region of Colombia were evaluated by an inhibition assay for their capacity to retard the growth of Plasmodium falciparum in vitro. The inhibitory activity was found to be independent of antibody activity. Furthermore, the degree of inhibition of parasite development was variable, depending on the parasite isolate used for the assay and the season of malaria transmission. We selected sera with high inhibitory activity and carried out partial analytical characterization by anion exchange fast protein liquid chromatography (FPLC) to identify the chemical nature of the inhibitory factor(s). The results suggested that the in vitro inhibitory activity might result from the additive effect of different molecules. It appears that these molecules could be non-specifically induced by stimulation of the immune system, they seem to play a role in the immunity to malaria.

Natural killer cell activity in experimental paracoccidioidomycosis of the Syrian hamster

The study evaluated the activity of NK cells during the course of experimental infection of hamsters with Paracoccidioides brasiliensis. Eigthy hamsters were infected with P. brasiliensis by intratesticular route and sacrificed at 24h, 48h, 96h, 1, 2, 4, 8 and 11 weeks of infection and compared to 40 noninfected hamsters employed as controls. These animals were submitted to the study of NK cytotoxic activity by a single-cell assay and humoral immune response by immunodiffusion and ELISA tests. The production of macrophage migration inhibitory factor in the presence of Phyto-hemagglutinin and P. brasiliensis antigen and histopathology of the lesions were evaluated at 1, 4, 8 and 11 weeks of infection. The infected animals displayed significantly high levels of NK activity during the four weeks of infection that decreased from the 8th week on when compared to controls. This impairment of NK activity was associated with depression of cell-mediated immune response and with increase in the extension of the histopathologic lesions. There was an inverse correlation between NK cell activity and specific antibody levels. The results suggest that after initial activation, NK cells were unable to control the fungus dissemination. The impairment of NK activity in the late stages of the infection might be related to immunoregulatory disturbances associated with paracoccidioidomycosis.

Clinical and laboratory findings in patients with dengue associated with hepatopathy

INTRODUCTION: Hepatic disorders caused by dengue infection may progress to severe manifestations, including mortality and morbidity. Cytokines are involved in it, such as the migration inhibitory factor of macrophages (MIF), tumor necrosis factor (TNF), natural killer cells (NK), B lymphocytes, and macrophages. METHODS: This study was carried out from January to April 2007 at a public hospital from the Federal University of Mato Grosso do Sul, Campo Grande, Brazil. Sixty-eight patients were studied concerning hepatic alterations, with 56 reported having classic dengue, 6 with hemorrhagic dengue grade I, and 6 with hemorrhagic dengue grade II. RESULTS: Among the 56 with classic dengue, 83.3% had aspartate aminotransferase (AST) alterations, and 69.6% had altered alanine aminotransferase (ALT). For those with hemorrhagic dengue grade I, 100% had AST alterations, and 83.3% had altered ALT. All the patients with hemorrhagic dengue grade II had AST and ALT alterations. AST variations reached 22.0 and 907.0, with an average value of 164.6. For ALT, we found variations between 25.0 and 867.0, with an average value of 166.07. There had been statistical significance between dengue clinical shapes and hepatic function markers. CONCLUSIONS: We conclude that the infection was predominant in adults, females, and in those with low income and education. The liver enzymes were of larger amount in hemorrhagic dengue, but there was weak statistical evidence of the clinical manifestations and transaminases. Major signs and clinical symptoms were fever, headache, myalgia, arthralgia, weakness, severe pain behind the eyes, and rashes.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Multifactorial control of water and saline intake: role of a2-adrenoceptors

Water and saline intake is controlled by several mechanisms activated during dehydration. Some mechanisms, such as the production of angiotensin II and unloading of cardiovascular receptors, activate both behaviors, while others, such as the increase in blood osmolality or sodium concentration, activate water, but inhibit saline intake. Aldosterone probably activates only saline intake. Clonidine, an a2-adrenergic agonist, inhibits water and saline intake induced by these mechanisms. One model to describe the interactions between these multiple mechanisms is a wire-block diagram, where the brain circuit that controls each intake is represented by a summing point of its respective inhibiting and activating factors. The a2-adrenoceptors constitute an inhibitory factor common to both summing points

Effect of chronic ethanol consumption in female rats subjected to experimental sepsis

The objective of this research was to evaluate the interference of ethanol consumption by female rats with cytokines involved in the sepsis process and its correlation with mortality, the main outcome of sepsis. Female Wistar rats in estrus phase were evaluated in three experiments. Experiment 1 (n=40) was performed to determine survival rates. Experiment 2 (n=69) was designed for biochemical analysis, measurement of cytokine and estrogen levels before and after sepsis, and experiment 3 (n=10) was performed to evaluate bacterial growth by colony counts of peritoneal fluid. In all experiments, treated animals were exposed to a 10% ethanol/water solution (v/v) as the single drinking source, while untreated animals were given tap water. After 4 weeks, sepsis was induced in the rats by ip injection of feces. In experiment 1, mortality in ethanol-exposed animals was delayed compared with those that drank water (48 h; P=0.0001). Experiment 2 showed increased tumor necrosis factor alpha (TNF-α) and decreased interleukin-6 (IL-6) and macrophage migration inhibitory factor in septic animals exposed to ethanol compared to septic animals not exposed. Sepsis also increased TNF-α and IL-6 levels in both ethanol- and water-exposed groups. Biochemical analysis showed higher creatinine, alanine aminotransferase and aspartate aminotransferase and decreased glucose levels in septic animals that were exposed to ethanol. In experiment 3, septic animals exposed to ethanol showed decreased numbers of colony-forming units than septic animals exposed to water. These results suggest that ethanol consumption delays the mortality of female rats in estrus phase after sepsis induction. Female characteristics, most probably sex hormones, may be involved in cytokine expression.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Clinical and immunological consequences of human T cell leukemia virus type-I and Schistosoma mansoni co-infection

Human T cell leukemia virus type-I (HTLV-I) infection is associated with spontaneous T cell activation and uncontrolled lymphocyte proliferation. An exacerbated type-1 immune response with production of pro-inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) is significantly higher in patients with myelopathy associated to HTLV-I than in HTLV-I asymptomatic carriers. In contrast with HTLV-I, a chronic Schistosoma mansoni infection is associated with a type-2 immune response with high levels of interleukin (IL-4, IL-5, and IL-10) and low levels of IFN-gamma. In this study, clinical and immunological consequences of the HTLV-I and S. mansoni infection were evaluated. The immune response in patients with schistosomiasis co-infected with HTLV-I showed low levels of IL-5 (p < 0.05) in peripheral blood mononuclear cells cultures stimulated with S. mansoni antigen (SWAP) and decreased SWAP-specific IgE levels when compared with patients with only schistosomiasis (p < 0.05). Liver fibrosis was mild in all HTLV-I co-infected patients. Immunological response was also compared in individuals who had only HTLV-I infection with those who were co-infected with HTLV-I and helminths (S. mansoni and Strongyloides stercoralis). In patients HTLV-I positive co-infected with helminths the IFN-gamma levels were lower than in individuals who had only HTLV-I. Moreover, there were fewer cells expressing IFN-gamma and more cells expressing IL-10 in individuals co-infected with HTLV-I and helminths. These dates indicate that HTLV-I infection decrease type 2-response and IgE synthesis and are inversely associated with the development of liver fibrosis. Moreover, helminths may protect HTLV-I infected patients to produce large quantities of pro-inflammatory cytokines such as IFN-gamma.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.