Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients.

Sm14 gene expression in different stages of the Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult worm

Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells.

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3% positive cells), alpha3 (93.3 ± 7.0%), alpha5 (50.4 ± 12.0%) and alpha6 (34.1 ± 4.9%) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0%) and fibronectin (40.0 ± 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4% vs DMSO: 70.7 ± 2.5%) while simultaneously reducing alpha5 (24.2 ± 19%) and alpha6 (14.3 ± 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

Differential expression of integrin subunits on adherent and nonadherent mast cells

Mast cell progenitors arise in bone marrow and then migrate to peripheral tissues where they mature. It is presumed that integrin receptors are involved in their migration and homing. In the present study, the expression of various integrin subunits was investigated in three systems of adherent and nonadherent mast cells. Mesentery mast cells, freshly isolated bone marrow-derived mast cells (BMMC) and RBL-2H3 cells grown attached to tissue culture flasks are all adherent mast cells and peritoneal mast cells, and cultured BMMC and RBL-2H3 cells grown in suspension represent nonadherent mast cell populations. Pure populations of mast cells were immunomagnetically isolated from bone marrow, mesentery and peritoneal lavage using the mast cell-specific monoclonal antibody AA4. By immunomicroscopy, we could demonstrate that all of these mast cells expressed alpha4, alpha5, alpha6, ß1 and ß7 integrin subunits. The expression of the alpha4 integrin subunit was 25% higher in freshly isolated mesentery mast cells and BMMC. Consistent with the results obtained by immunomicroscopy, mesentery mast cells expressed 65% more mRNA for the alpha4 integrin subunit than peritoneal mast cells. In vitro studies were also conducted using the rat mast cell line RBL-2H3. RBL-2H3 cells grown attached to the tissue culture flasks or as suspension cultures expressed the same integrin subunits identified in bone marrow, mesenteric and peritoneal mast cells ex vivo. Similarly, the expression of alpha4 integrin was higher in adherent cells. Therefore, alpha4 integrins may play a critical role in the anchorage of mast cells to the extracellular matrix in bone marrow and in peripheral tissues.

Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ± 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol™ and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

Selegiline increases heme oxygenase-1 expression and the cytotoxicity produced by dopamine treatment of neuroblastoma SK-N-SH cells

Increased dopamine catabolism may be associated with oxidative stress and neuronal cell death in Parkinson's disease. The present study was carried out to examine the effect of dopamine on the expression of heme oxygenase-1 and -2 (HO-1 and HO-2) in human neuroblastomas (SK-N-SH cell line) and the effects of selegiline and antioxidants on this expression. Cells were kept with close control of pH and were incubated with varying concentrations of dopamine (0.1-100 µM) for 24 h. HO-1 and HO-2 cDNA probes were prepared by reverse transcription-polymerase chain reaction amplification. The mRNA expression of HO-1 and HO-2 was measured by Northern blot analysis. The levels of HO-1 mRNA increased after dopamine treatment, in a dose-dependent manner, in all cell lines studied, whereas levels of the two HO-2 transcripts did not. The HO-1 and HO-2 protein expression was analyzed by Western blotting. HO-1 protein was undetectable in untreated SK-N-SH cells and increased after treatment with dopamine. In contrast, the HO-2 protein (36 kDa) was detected in untreated cells and the levels did not change as a result of treatment. alpha-Tocopherol (10-100 µM) and ascorbic acid (100 µM) did not attenuate the effects of dopamine. Selegiline (10 µM) produced significant increase (P < 0.01) in the induction of HO-1 by dopamine (more than six times the control values). The increased expression of HO-1 following dopamine treatment indicates that dopamine produces oxidative stress in this cell line.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknüllt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55°C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Low expression of APAF-1XL in acute myeloid leukemia may be associated with the failure of remission induction therapy

Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65% of the samples (group 1), while 35% of the samples expressed primarily APAF-1LN (group 2). Only 46% of the patients presented complete remission in response to remission induction therapy (represented by less than 5% marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6% did not attain complete remission (only 1 case from group 1), and 32.4% of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.

Effects of exercise training on atrophy gene expression in skeletal muscle of mice with chronic allergic lung inflammation

We evaluated the effects of chronic allergic airway inflammation and of treadmill training (12 weeks) of low and moderate intensity on muscle fiber cross-sectional area and mRNA levels of atrogin-1 and MuRF1 in the mouse tibialis anterior muscle. Six 4-month-old male BALB/c mice (28.5 ± 0.8 g) per group were examined: 1) control, non-sensitized and non-trained (C); 2) ovalbumin sensitized (OA, 20 µg per mouse); 3) non-sensitized and trained at 50% maximum speed _ low intensity (PT50%); 4) non-sensitized and trained at 75% maximum speed _ moderate intensity (PT75%); 5) OA-sensitized and trained at 50% (OA+PT50%), 6) OA-sensitized and trained at 75% (OA+PT75%). There was no difference in muscle fiber cross-sectional area among groups and no difference in atrogin-1 and MuRF1 expression between C and OA groups. All exercised groups showed significantly decreased expression of atrogin-1 compared to C (1.01 ± 0.2-fold): PT50% = 0.71 ± 0.12-fold; OA+PT50% = 0.74 ± 0.03-fold; PT75% = 0.71 ± 0.09-fold; OA+PT75% = 0.74 ± 0.09-fold. Similarly significant results were obtained regarding MuRF1 gene expression compared to C (1.01 ± 0.23-fold): PT50% = 0.53 ± 0.20-fold; OA+PT50% = 0.55 ± 0.11-fold; PT75% = 0.35 ± 0.15-fold; OA+PT75% = 0.37 ± 0.08-fold. A short period of OA did not induce skeletal muscle atrophy in the mouse tibialis anterior muscle and aerobic training at low and moderate intensity negatively regulates the atrophy pathway in skeletal muscle of healthy mice or mice with allergic lung inflammation.

Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection

Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKβ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100% of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.